Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

A monoclonal antibody reactive with an activated ras protein expressing valine at position 12

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000-Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown to contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.     

Activated c-N-ras in radiation-induced acute nonlymphocytic leukemia: twelfth codon aspartic acid

We describe the detection and characterization of an activated c-N-ras allele from a gamma-radiation-induced canine acute nonlymphocytic leukemia (ANLL). The activated allele was detected by use of the NIH3T3 transfection/transformation assay. The leukemia DNA had a transforming activity of 0.0125 foci/microgram. By the use of a double anti-ras antibody enzyme-linked immunoblot assay, we have dissected the lesion within the activated c-N-ras allele. Aspartic acid has been substituted for the normal glycine at position 12 in the activated p21c-N-ras. The expression of the mutant p21ras has also been detected in an in vivo passage of the radiation-induced canine ANLL from which the activated c-N-ras allele was isolated. We have demonstrated sufficient homology between canine c-N-ras genes and the human cDNA c-N-ras clone, p6a1, that allows this probe to be used in Southern blotting of canine tissues. In addition, anti-ras antibodies generated against both murine and human ras antigens are capable of detecting canine p21ras species.     

Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells

We report the isolation and characterization of two human liver cDNA clones, HUG-Br1 and HUG-Br2; each encodes a UDP-glucuronosyltransferase enzyme which glucuronidates bilirubin IX alpha to form both the IX alpha C8 and IX alpha C12 monoconjugates and a diconjugate. HUG-Br1 cDNA (2351 base pairs) and HUG-Br2 cDNA (2368 base pairs) encode proteins with 533 and 534 amino acid residues, respectively, with a typical membrane-insertion signal peptide, membrane-spanning domain, and 3 or 5 potential asparagine-linked glycosylation sites. At the nucleic acid and deduced amino acid sequence levels the two clones are 82% similar overall, 66% similar in the amino termini, and identical after codon 287, thus encoding proteins with the same carboxyl terminus. The mRNA encoding HUG-Br1 is of high abundance, and the one encoding HUG-Br2 is of low abundance; both are 2.6 kilobases in length. Both messages (2.6 kilobases) were present in the explanted liver of a Type I Crigler-Najjar patient, although the level for that of HUG-Br1 was reduced 4.5-fold. Northern blot analysis of poly(A)+ RNA isolated from the liver of an untreated and a phenobarbital-treated Erythrocebus patas monkey with 5'-specific probes for each clone indicated that the HUG-Br2-encoded message is induced two fold, but that for HUG-Br1 is not. These data indicate that bilirubin is glucuronidated by at least two different proteins, most likely present in very different amounts. These cDNAs which encode functional bilirubin UDP-glucuronosyltransferases will allow the isolation of an appropriate gene to develop a gene therapy model for patients which have the totally deficient trait.     

Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines

Suppression of PKC activity can selectively induce apoptosis in cells expressing a constitutively activated p21Ras protein. We demonstrate that continued expression of p21Ras activity is required in PKC-mediated apoptosis because farnesyltransferase inhibitors abrogated the loss of viability in p21Ras-transformed cells occurring following PKC inhibition. Studies utilizing gene transfer or viral vectors demonstrate that transient expression of oncogenic p21Ras activity is sufficient for induction of apoptosis by PKC inhibition, whereas physiologic activation of p21Ras by growth factor is not sufficient to induce apoptosis. Mechanistically, the p21Ras-mediated apoptosis induced by PKC inhibition is dependent upon mitochondrial dysregulation, with a concurrent loss of mitochondrial membrane potential (psim). Cyclosporine A, which prevented the loss of psim, also inhibited HMG-induced DNA fragmentation in cells expressing an activated p21Ras. Induction of apoptosis by PKC inhibition in human tumors with oncogenic p21Ras mutations was demonstrated. Inhibition of PKC caused increased apoptosis in MIA-PaCa-2, a human pancreatic tumor line containing a mutated Ki-ras allele, when compared to HS766T, a human pancreatic tumor line with normal Ki-ras alleles. Furthermore, PKC inhibition induced apoptosis in HCT116, a human colorectal tumor line containing an oncogenic Ki-ras allele but not in a subline (Hke3) in which the mutated Ki-ras allele had been disrupted. The PKC inhibitor 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG), significantly reduced p21Ras-mediated tumor growth in vivo in a nude mouse MIA-PaCa-2 xenograft model. Collectively these studies suggest the therapeutic feasibility of targeting PKC activity in tumors expressing an activated p21Ras oncoprotein.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion.

The rat neu gene, which encodes a receptor-like protein homologous to the epidermal growth factor receptor, is frequently activated by a point mutation altering a valine residue to a glutamic acid residue in its predicted transmembrane domain. Additional point mutations have been constructed in a normal neu cDNA at and around amino acid position 664, the site of the naturally arising mutation. A mutation which causes a substitution of a glutamine residue for the normal valine at residue 664 leads to full oncogenic activation of the neu gene, but five other substitutions do not. Substituted glutamic acid residues at amino acid positions 663 or 665 do not activate the neu gene. Thus only a few specific residues at amino acid residue 664 can activate the oncogenic potential of the neu gene. Deletion of sequences of the transforming neu gene demonstrates that no more than 420 amino acids of the 1260 encoded by the gene are required for full transforming function. Mutagenesis of the transforming clone demonstrates a correlation between transforming activity and tyrosine kinase activity. These data indicate that the activating point mutation induces transformation through (or together with) the activities of the tyrosine kinase.     

cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B

Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP-A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled.