Violet Daniel

Role of Oxidants and Antioxidants in the Induction of AP-1, NF-κB, and Glutathione S-Transferase Gene Expression

Transcription factors AP-1 and NF-κB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of ·OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of ·OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-κB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-κB activities are induced by oxygen radicals.

Bacteriophage Induced Transfer RNA in Escherichia coli

The events taking place after a viral nucleic acid enters a susceptible host cell depend on the specific viral system. In the case of the DNA-containing viruses (1), the nucleic acid serves as a template both for its own replication and for the transcription of viral specific mRNA which is then translated into viral proteins. It has been generally thought that the various components of the host translational system remain unaltered and are utilized for the synthesis of viral proteins; the viral mRNA becomes attached to the preexisting host cell ribosomes, and viral polypeptides are then synthesized by the preexisting host cell tRNA and aminoacyl-tRNA synthetases. This is a rather simplified presentation of the events taking place during synthesis of viral proteins. Recent experiments show that the picture may be more complicated, and, at least in the case of some viruses, a more intricate translational mechanism is involved. Studies from several laboratories show that mammalian and bacterial viruses may induce changes in the translational mechanism of their host cells. These changes were observed in the tRNA, in the enzymes modifying the tRNA (such as tRNA methylases and tRNA thiolases), or in the aminoacyl-tRNA synthetases. The evidence for changes in the translation mechanism which follow virus infection is summarized below. Several modifications of tRNA induced by virus infection have been observed. Thus, the chromatographic profile of leucine tRNA in Escherichia coli is altered after the bacterial cells have been infected with T2 bacterio-

Sequence analysis suggests a recent duplication of the growth hormone-encoding gene in Tilapia nilotica

The sequence of two growth hormone(GH)-encoding genes from tilapia fish (Tilapia nilotica) is reported. Our data indicate that the presence of two GH in the tilapia genome is a consequence of a relatively recent duplication event. The two genes are highly homologous, having a similar intron (five)/exon (six) arrangement, and both encode an identical polypeptide. Sequence similarity extends up to bp -628 upstream to the transcription start point, after which the sequences of the two genes are not related to each other. The presence of two GH in the tilapia genome is supported both by the nucleotide sequence and by genomic DNA blot hybridization analysis. Tilapias, like salmonids, contain an extra intron compared with the mammalian GH structure. We suggest that within the superorder Teleostei, the insertion of intron 5 into GH took place after the evolutionary separation of Cyprinoidea, but before Isospondyli (salmonids) and Acanthopterygii (tilapias) were separated. Thus, the additional intron which is probably present in many teleost fish GH may provide an excellent natural marker for evolution and classification studies.

Structure and sequence of the growth hormone-encoding gene from Tilapia nilotica

We report here the nucleotide (nt) sequence of the growth hormone (GH)-encoding gene (GH) of the tilapia fish (Tilapia nilotica). The T. nilotica GH gene, similar to that of the salmonidae fish, Atlantic salmon and rainbow trout, contains six exons and five introns. However, despite the presence of an additional intron (intron V), the size of the primary transcript of T. nilotica GH (1666 nt) is significantly shorter than that of all other currently characterized fish GH genes. Comparison of sequences upstream from the transcription start point of the tilapia, carp, rainbow trout and Atlantic salmon GH genes shows a region of high homology preceding the typical TATA box. This homology does not seem to extend to the regions further upstream of the compared fish GH genes and is not observed to be present in the corresponding region of the mammalian GH genes. A sequences search for putative DNA-binding domains for transcription factors shows the presence of short nt stretches similar to those considered to be involved in the tissue-specific expression of mammalian GH genes.

Amino acid acceptor activity of bacteriophage T4 transfer RNA

In a recent study we have shown that T4 bacteriophage infection of E. coli B cells induces the formation of 4s RNA molecules that specifically hybridize with T4 DNA [1,2] . Moreover, the T4 4s RNA extracted from the hybrid was found to contain pseudouridylic acid [2]. Since IL UMP is believed to be present predominantly in transfer RNA, its presence in a 4S RNA molecule coded for by the T4 genome suggested that at least some of this RNA may have amino acid acceptor function. To test this hypothesis a method was devised to recover intact, biologically active tRNA from tRNADNA hybrids [3]. We recently reported that the T4 4S RNA preparation extracted from a hybrid with T4 DNA has amino acid acceptor capacity [4]. In addition, Weiss et al. [S] showed that leucyl-tRNA isolated from TCinfected cells will hybridize with T4 DNA. This report describes the method used for the isolation of biologically active T4 tRNA from its hybrid with T4 DNA.

Biosynthesis of pseudouridine in the in vitro transcribed tRNATyr precursor

The biosynthesis of a tRNA molecule in prokaryotes starts with the transcription of an RNA precursor which is converted to tRNA via a multistep process involving nucleolytic cleavages, which progressively reduce its length, and the modification of several nucleosides along the sequence [l] . Since the intracellular concentration of the maturation intermediates is generally very low, the study of tRNA biosynthesis has been greatly facilitatea by the isolation of mutants altered in some specific steps of the biosynthetic pathway, with consequent accumulation of tRNA precursors [2-61. In a previous study [3,7] it has been reported that hisT mutants of Salmonella typhimurium lack an enzyme responsible for the biosynthesis of pseudouridine in the anticodon region of several tRNA species. tRNA Extracted from hisT mutants has been used as substrate for in vitro biosynthesis of pseudouridine and an assay has been set up, which is based on the release of tritium from [ 5-3H] uridine-labeled hisT tRNA, upon conversion of uridine into pseudouridine [ 71.

Transfer of soluble polynucleotides to microsomal RNA

Abstract Detergent-treated microsomes prepared from rat liver, were found to be active in the transfer of soluble polynucleotides to ribonucleic acid of microsomes. The reaction was dependent on the presence of adenosine triphosphate and upon an energy generating system. The extent of transfer of isotope from [32P]soluble ribonucleic acid, freed from attached amino acids, to microsomal ribonucleic acid was not affected by the addition of soluble enzymes (105,000 × g supernatant) and an amino acid mixture. Transfer of radioactivity to microsomal ribonucleic acid was also found when soluble ribonucleic acid, labelled with [14C]adenosine monophosphate in the terminal position, was used. When labelled microsomal ribonucleic acid isolated from the reaction mixture was hydrolysed with alkali, 63% of the recovered radioactivity was found in adenosine monophosphate and about 37% in the adenosine fraction. By contrast, after alkaline hydrolysis of the soluble ribonucleic acid terminally labelled with [14C]adenosine monophosphate 93% of the radioactivity was found in the adenosine fraction.

Mapping of transcription units in the bacteriophage T4 tRNA gene cluster

Abstract The transfer RNA gene cluster of bacteriophage T4 (about 1600 base-pairs long) is comprised of ten genes that code for eight tRNA and two other stable RNA species. It is arranged in two tight subclusters separated by a spacer of 600 base-pairs. We studied the transcription of these genes in vitro by using DNA templates derived from a series of T4 mutants partially deleted within the tRNA genetic region. The mixtures of primary products made by RNA polymerase on the different T4 DNA templates were fractionated by gel electrophoresis into discrete RNA species. Wildtype and deletion-specific transcripts of the tRNA gene cluster were isolated and their sizes and stable RNA sequence composition were determined. Three primary transcripts of the intact tRNA operon, one major and two minor species, were identified. The major RNA species measured about 3000 nucleotides and harboured the sequences of both subclusters of stable RNA genes. The two minor RNA species (2300 and 1700 nucleotides, respectively) lacked the sequences of the promoterdistal gene subcluster. The existence of a fourth minor primary transcript (about 3500 nucleotides) that contains the sequences of both gene subclusters is inferred from the results. On the basis of these data and the location of a promoter for the T4 tRNA gene cluster by Fukada et al. (1980b), we suggest two alternative schemes for transcriptional organization of the T4 tRNA genes. Both schemes envisage a major transcription unit defined by a promoter 1000 base-pairs upstream, and a terminator 300 base-pairs downstream from the tRNA genes. The existence of weak terminator(s) in the spacer region is also proposed. Primary transcripts of the tRNA operon were processed with S-100 extracts of Escherichia coli and nine T4-specific stable RNAs were identified among the products. This indicates that E. coli contains all the enzymes necessary for the processing of polycistronic T4 tRNA precursors. The in vitro synthesized primary transcripts provide useful substrates for the study of processing enzymes involved in the early steps of tRNA maturation.

Effect of N6,O2'-dibutyryl cyclic AMP treatment of lymphoma cells on the translation of globin mRNA by cell-free extracts.

The presence of CAMP-dependent protein kinase in many tissues in which CAMP functions as a second messenger has led Kuo and Greengard [ 1 ] to propose that CAMP may act in eukaryotes exclusively by activating a protein phosphokinase. According to this hypothesis the biological activity of cellular proteins is modified via specific phosphorylations, a mechanism demonstrated for the regulatory effects of CAMP in glycogen metabolism and in lipolysis [2-51. Recent studies bring evidence for a possible role of protein kinase also in the regulation of cell growth [6] and the induction of specific enzymes by CAMP [7,8]. Although the site of regulation by CAMP was not definitively demonstrated, experiments indicate that the cyclic nucleotide stimulates the phosphorylation of both nuclear [9,10] and ribosomal proteins [ 1 l-141. The fact that the eukaryotic ribosomal proteins are phosphorylated in vivo and in vitro by CAMP-dependent protein kinase implies that the function of the ribosomes might be regulated by the reversible phosphorylation of their proteins. In vitro studies in a cell-free protein synthesizing system did not, however, bring evidence as to a functional role for ribosomal protein phosphorylation [ 151. As an approach to the study of the effects of CAMP on the protein synthesizing machinery of an eukaryotic cell we have used a mouse lymphoma cell line in which db CAMP induces growth inhibition and

Relative stabilities of RNA/DNA hybrids: Effect of RNA chain length in competitive hybridization

Abstract Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA Tyr -like sequences transcribed in vitro on φ80p su 3 + bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA Tyr hybridization revealed that the in vitro -synthesized RNA contained significant amounts of tRNA Tyr ; these sequences could not, however, be detected by the direct competition method in which labelled in vitro -synthesized RNA competes with E. coli tRNA Tyr for hybridization to φ80p su 3 + DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro -transcribed tRNA Tyr -like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA Tyr from its hybrids with φ80p su 3 + DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA Tyr .