Gene organization of the transforming region of adenovirus type 7 DNA

The sequence of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has been determined. This part of the Ad7 viral genome encompasses early region E1 which has been shown to be involved in the process of cell transformation in vitro (Dijkema et al., 1979). From the nucleotide sequence and determined coordinates of the E1 mRNAs, we are able to predict the primary structure of the polypeptides encoded by the transforming region of Ad7. The organization of the E1 region of Ad7 and of other adenovirus serotypes (Bos et al. 1981) leads to the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large E1b-specific tumor antigens of adenovirus types 12, 7 and 5 may explain the differences in oncogenicity of these viruses.     

Size and structure of the hydrophobic low molecular weight surfactant-associated polypeptide

The most abundant low molecular weight protein of pulmonary surfactant has unusual properties. Its primary structure has now been determined by analysis at the protein level. The highly hydrophobic polypeptide is resistant to cleavage with proteolytic enzymes, but it was possible to generate fragments by limited cleavage with concentrated HCl or with sodium in liquid ammonia. Acid hydrolysis of the peptide required exceptional conditions for release of all residues. The N-terminus is heterogeneous, and in its longest form the primary structure consists of 35 residues. This analysis establishes that the size of the major native hydrophobic surfactant polypeptide is considerably smaller than previously proposed. Biological effects of the polypeptide recombined with phospholipids are confirmed in vitro by using a pulsating bubble system and in vivo by using premature newborn rabbits. The molecule has branched-chain amino acid residues at about two-thirds of all positions and lacks nine types of residue. The middle third is composed entirely of hydrophobic residues, and fragments from this part are sparingly soluble even in organic solvents. The hydrophobic region is preceded by a more hydrophilic, N-terminal segment. Thus, the molecule has two contrasting parts, like a detergent, which may explain its essential role in the pulmonary surfactant system.     

Identification of a low molecular weight form of epithelial transforming growth factor

We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.     

A novel metal-free low molecular weight superoxide dismutase mimic

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.     

Production of a low molecular weight growth inhibitory factor by adenovirus 12-transformed cells.

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.     

A low molecular weight DNA polymerase from wheat embryos

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase C_I) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase C_I did not inhibit animal DNA polymerases and or wheat DNA polymerase A, whereas wheat DNA polymerases C_II and B were much less affected than the C_I enzyme. Several properties of enzyme C_I were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase C_I while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase C_I can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase . The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase , an enzyme believed to be involved in nuclear DNA repair.     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

The sequence of the 3'' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.     

Isolation of a 32P-labeled polypeptide of low molecular weight from phosphorylated human erythrocyte membranes.

The incorporation of 32P into well washed human erythrocyte membranes was studied in a medium containing [gamma-32P]ATP, Mg2+, and EGTA. Following phosphorylation, the membranes were completely solubilized in 1% sodium dodecyl sulfate and subjected to gel electrophoresis in dodecyl sulfate polyacrylamide. A large incorporation of radioactivity was observed in a band which migrated faster than component 7 (nomenclature of T. L. Steck, (1972), J. Mol. Biol. 66, 295) but slower than the bromophenol blue tracking dye, and did not stain with Coomassie Blue. Isolation of this band by preparative gel electrophoresis revealed that 41% of the radioactivity was associated with a 32P-labeled polypeptide. This polypeptide was further purified by gel chromatography on Sephadex LH-20 in chloroform-methanol-HCl, and Bio-Gel A 1.5m in dodecyl sulfate. Its amino acid composition is characterized by a high content of acidic residues. The calculated minimal molecular weight is 15084. Based upon the recovery of amino acids, the polypeptide fraction comprises at least 1.8% by weight of the total erythrocyte membrane proteins. An apparent molecular weight of 15000 was estimated by gel chromatography in dodecyl sulfate, while a range of 14000-16000 was estimated by electrophoresis in dodecyl sulfate polyacrylamide. The state of phosphorylation of this peptide may reflect a physiological function in the intact red cell.     

Phosphorylation of a low molecular weight polypeptide in beef heart Na plus, K plus-ATPase preparations.

Sodium dodecyl sulfate-polyacrylamide gel profiles of a NaI-treated beef heart Na⁺,K⁺-ATPase preparation revealed the presence of two protein kinase substrates of low molecular weight, whereas a more purified citrate beef heart Na⁺,K⁺-ATPase preparation contained one low molecular weight polypeptide substrate. This enzyme preparation was phosphorylated in the presence of protein kinase, and phosphorylation was inhibited by protein kinase inhibitor. The phosphorylated product was identified as a phosphoester. Half maximal stimulation of protein kinase-catalyzed phosphorylation occurred at approximately 9 × 10^?8m cyclic AMP. The low molecular weight (11,700) protein kinase substrate present in the heart preparations was eluted from polyacrylamide slab gels. The polypeptide fraction was reelectrophoresed and the polypeptide was removed from the gels, hydrolyzed, and analyzed for amino acid content. This polypeptide was different from other low molecular weight protein kinase substrates including troponin components, myosin light chains, and histones and is most likely of plasma membrane origin.     

The low molecular weight of RNAs of adenovirus 2-infected cells

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA_I, and the minor species is 5.2 S RNA or virus-associated RNA_II. Virus-associated RNA_I occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA_II is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the r strand near position 30 of the adenovirus type 2 physical map, and are separated by a spacer of about 75 base-pairs.     

A novel low molecular weight phospholipase D from Streptomyces sp. CS684

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.     

Structural characterization of the proteins encoded by adenovirus early region 2A

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.     

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.     

Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein

The 5′ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M_r protein, linked to a small [³²P]oligonucleotide, was isolated after DNase digestion of uniformly ³²P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [³²P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5′ ends of Ad5 DNA); (3) prior treatment of the [³²P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of O-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mm-NaOH at 70 °C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad5 DNA under these conditions we were able to label the 5′ termini with ³²P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5′ mononucleotides yielded [³²P]dCMP. We conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the β-OH of a serine residue of the protein and the 5′-OH of the terminal deoxycytidine residue of the DNA.