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PubMed1983

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator.

Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

Escherichia coli↗

PubMed1987

A complex array of sequences enhances ribosomal transcription in Xenopus laevis.

The ribosomal DNA spacer in Xenopus laevis was shown in previous studies to be involved in regulating the expression of the ribosomal genes. Here transcription enhancement by this spacer has been studied in some detail, to fully identify the sequences involved and to determine their relative importance in this phenomenon. It is shown that the 60/81 base-pair (bp) repeats, which were reported to be enhancer elements, act as part of a mode of enhancement whose effect is amplified by the spacer promoters or Bam islands. The "Bam super repeat", a combination of spacer promoter and 60/81 bp elements, is the major enhancer unit. Within a Bam super repeat, a near linear correlation between the number of 60/81 bp elements and enhancer activity is observed. Thus, there is no significant co-operativity in the binding of transcription factors to an array of these elements. Multiple Bam super repeats do not act additively and may actually interfere with each others action. Surprisingly this effect is observed both in the presence and absence of active spacer promoters. Sequences between the 3' end of the 28 S coding region and the first spacer promoter may also be involved in enhancement but only in a very minor fashion. In confirmation of recent studies, the presence of the unique ribosomal termination sequence, 213 bp upstream from the pre-rRNA initiation site, is essential for efficient promotion, as deletion of this sequence virtually abolishes pre-rRNA- transcription. These data are discussed in terms of the possible mechanisms of transcription enhancement.

Animals↗

PubMed1998

Some traces of hidden codes.

Recent results in comparative genetics reveal processes of neutral evolution that are reminiscent of neutral codon substitutions, although they operate on novel kinds of sequences and molecular structures. This suggests that in addition to the genetic code, previously unrecognized "degenerate" codes might govern molecular interactions at other levels of biological information. If they exist, such multiple degenerate codes cannot be accounted for by the two concepts usually placed at the heart of molecular biology, i.e. molecular cascades and gene networks. This article reports on some representative examples of putative degenerate codes involving three fundamental levels of biological regulation, i.e. transcription, post-transcriptional regulation and signal transduction. From these examples we suggest that degenerate codes are organized hierarchically and that the concept of neutral evolution can be generalized to all kinds of molecular interactions. In addition, a case of functional evolution is interpreted as the emergence of novel, possibly degenerate codes. Comparative genetics and molecular embryology will be instrumental in testing the existence of multiple degenerate codes and hence, in unraveling the causes of evolution.

Animals↗

PubMed2001

Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements.

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

Bacterial Proteins↗

PubMed1993

The nuclear-encoded polypeptide Cfo-II from spinach is a real, ninth subunit of chloroplast ATP synthase.

Proton-translocating F-ATP synthases from chloroplasts contain a nuclear-coded subunit, CFo-II, that lacks an equivalent in the corresponding E. coli complex. Three recombinant phages that code for the entire precursor of this subunit have been isolated from lambda gt11 cDNA expression libraries made from polyadenylated spinach RNA using a two-step strategy. The reading frame of 222 amino acid residues includes 147 residues for the mature protein (M(r) 16.5 kDa) and a transit sequence of 75 residues (M(r) 8.0 kDa). Secondary structure predictions indicate a bitopic protein, anchored by a single N-terminal transmembrane segment and a C-terminal hydrophilic region that probably reaches into CF1. CFo-II precursor made in vitro can be imported into isolated, intact chloroplasts and assembled into ATP synthase. This protein is a real subunit of the plastid enzyme and a distinctive characteristic of ATP synthases involved in photosynthetic processes. Unique features are (i) that the gene for CFo-II (atpG) appears to be a duplication of atpF encoding CFo-I, the homologues of the genes for subunits b' and b in photosynthetic bacteria, (ii) that it represents the first instance that one copy of the various duplicated loci found in plastid chromosomes has been phylogenetically translocated to the nucleus, and (iii) that it operates with a bipartite (import/thylakoid-targeting) transit peptide but without an intermediate cleavage site for the stroma protease, suggestive of a way of membrane integration different from that of its plastome-encoded counterpart CFo-I. With these data, the first complete sequence for a chloroplast ATP synthase of a higher plant (spinach) is available.

Amino Acid Sequence↗

PubMed1994

A corolla- and carpel-abundant, non-specific lipid transfer protein gene is expressed in the epidermis and parenchyma of Gerbera hybrida var. Regina (Compositae).

We are examining the floral organ differentiation in Compositae by isolating and characterizing corolla abundant genes. Differential screening of a cDNA library made from the ray floret corolla of Gerbera hybrida var. Regina revealed an abundant cDNA clone which is expressed in the corolla but not in leaves. This cDNA (gltp1) codes for a polypeptide similar to non-specific lipid transfer proteins of the plants. The gltp1 gene is expressed only in the corolla and carpels and is developmentally regulated during corolla development. The gltp1 mRNA accumulates both in epidermal cell layers and in the mesophyll of the corolla. In the stylar part of the carpel, the gltp1 mRNA can be detected in the epidermal and in parenchymal cells but not in the transmitting tissue. Analogous patterns of gltp1 expression in the corolla and carpel may indicate that similar genetic programmes operates during the development of these two tissues.

Amino Acid Sequence↗

PubMed2003

The web server of IBM's Bioinformatics and Pattern Discovery group.

We herein present and discuss the services and content which are available on the web server of IBM's Bioinformatics and Pattern Discovery group. The server is operational around the clock and provides access to a variety of methods that have been published by the group's members and collaborators. The available tools correspond to applications ranging from the discovery of patterns in streams of events and the computation of multiple sequence alignments, to the discovery of genes in nucleic acid sequences and the interactive annotation of amino acid sequences. Additionally, annotations for more than 70 archaeal, bacterial, eukaryotic and viral genomes are available on-line and can be searched interactively. The tools and code bundles can be accessed beginning at http://cbcsrv.watson.ibm.com/Tspd.html whereas the genomics annotations are available at http://cbcsrv.watson.ibm.com/Annotations/.

Computational Biology↗

PubMed1998

Tetracycline-regulated suppression of amber codons in mammalian cells.

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Acylation↗

PubMed1994

Ventral tegmental area (VTA) injections of tyrosine hydroxylase phosphorothioate antisense oligonucleotide suppress operant behavior in rats.

Dopaminergic A 10 neurons are known to be the essential part of the brain reinforcement system. An antisense oligonucleotide corresponding to the start coding region of rat tyrosine hydroxylase (TH) mRNA, the transcriptional message of the rate limiting enzyme in the metabolic pathway leading to catecholamine synthesis, was constructed and injected into the ventral tegmental area (VTA). 36 h after injection operant behavior was markedly reduced, and this suppression was fully reversed within 5 days following the antisense injection. Accordingly, TH immunoreactivity in the VTA was reduced in comparison to control experiments using mixed bases oligonucleotides. Our results demonstrate that highly specific inhibition of TH expression can be accomplished in the intact mesolimbic system by antisense treatment, thus providing a novel tool for studies on motivational processes in vivo.

Animals↗

PubMed1983

Prostaglandins and gene action: possible relevance to the effect of PG system on leukocyte alkaline phosphatase enzyme activity.

Prostaglandin E2, colchicine and imidazole, known to enhance and inhibit thromboxane A2 (TxA2) synthesis respectively and chloroquine, a PGE1 synthesis enhancer and PGE2 antagonist can alter the leukocyte alkaline phosphatase (LAP) enzyme activity. Thromboxane A2 and other related prostaglandins (PGs) are known to bind to DNA and thus possibly regulate DNA action. It is proposed here that prostaglandins are able to modify LAP activity by their action on the DNA and thus the Gene that codes for LAP. This hypothesis envisages that normally there are specific receptors on the genes for PGs. Taking LAP enzyme system as the model, it is proposed that the affinity of TxA1, TxA2, PGE2 and PGE1 to the operator is more than to that of the repressor. But when the levels of TxA2, PGE2 and PGE1 are in excess, all the receptors on the operator are not only completely occupied but they are also able to bind to repressor in sufficient amounts to transiently switch off the LAP synthesis by the structural gene. It is further envisaged that the affinity of the PG receptors on the operator and repressor systems of the operon is greatest for TxA1 and least for PGE2 (TxA1 greater than TxA2 greater than PGE1 greater than PGE2). This hypothesis though simply in its model explains all the variables obtained in our studies on the effect of PGs on the LAP activity. This concept by itself or a suitable modification, may explain the role of PGs in the pathogenesis of cancer. It will be interesting to study, in the light of this hypothesis, the role of the PG system on DNA repair mechanism, m-RNA and t-RNA synthesis and gene action in several systems.

Alkaline Phosphatase↗

PubMed2002

Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

3' Untranslated Regions↗

PubMed1984

Polymorphic sites and the mechanism of evolution in human mitochondrial DNA.

Twelve restriction enzymes were used to screen for the presence or absence of cleavage sites at 441 locations in the mitochondrial DNA of 112 humans from four continents. Cleavage maps were constructed by comparison of DNA fragment sizes with those expected from the published sequence for one human mtDNA. One hundred and sixty-three of the sites were polymorphic, i.e., present in some individuals but absent from others, 278 sites being invariant. These polymorphisms probably result from single base substitutions and occur in all functional regions of the genome.--In 77 cases, it was possible to specify the exact nature and location (within a restriction site) of the mutation responsible for the absence of a restriction site in a known human mtDNA sequence and its presence in another human mtDNA. Fifty-two of these 77 gain mutations occur in genes coding for proteins, 34 being silent and 18 causing amino acid replacements; moreover, nine of the replacements are radical.--Notable also is the anomalous ratio of transitions to transversions required to account for these 77 restriction site differences between the known human mtDNA sequences and other human mtDNAs. This ratio is lower for most groups of restriction sites than has been reported from sequence comparisons of limited parts of the mtDNA genome in closely related mammals, perhaps indicating a special functional role or sensitivity to mutagenesis for palindromic regions containing high levels of guanine and cytosine.--From the genomic distribution of the 163 polymorphic sites, it is inferred that the level of point mutational variability in tRNA and rRNA genes is nearly as high as in protein-coding genes but lower than in noncoding mtDNA. Thus, the functional constraints operating on components of the protein-synthetic apparatus may be lower for mitochondria than for other systems. Furthermore, the mitochondrial genes for tRNAs that recognize four codons are more variable than those recognizing only two codons.--Among the more variable of the human mitochondrial genes coding for proteins is that for subunit 2 of cytochrome oxidase; this polypeptide appears to have been evolving about five times faster in primates than in other mammals. Cytochrome c, a nuclearly encoded protein that interacts directly with the oxidase 2 subunit in electron transport, has also evolved faster in primates than in rodents or ungulates. This example, along with that for the mitochondrial rRNA genes and the nuclear genes coding for mitochondrial ribosomal proteins, provides evidence for coevolution between specific nuclear and mitochondrial genes.

Base Sequence↗

PubMed1987

The yeast tRNA(Phe) gene family: structures and transcriptional activities reveal member differences not explained by intragenic promoters.

Several cloned members of the yeast tRNA(Phe) gene family were transcribed in vitro using a HeLa extract and a yeast extract. The optimum DNA concentration was determined and kinetic experiments were performed for each clone to compare transcription levels. Both extract systems were able to splice the intervening sequence, but only the yeast extract produced the mature product. Some genes were not transcribed with the homologous system while they were transcribed with the HeLa extract, suggesting a control mechanism that is not operating in the heterologous system. Competition experiments demonstrated that the intragenic promoters of the inactive genes were able to bind transcription factor(s), but not as efficiently as active genes. This binding was not so strong when using linear DNA and was dependent on the presence of the 3' intragenic control region. DNA sequencing and computer analysis indicated the presence of short conserved sequences upstream from the genes. These sequences, which are not related to the intragenic promoters, are direct repeats of part of the 3' coding region in those genes that are transcribed in the homologous system. The relevance of these sequences on homologous transcription in vitro remains to be established.

Base Sequence↗

PubMed1984

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.

Aspergillus nidulans↗

PubMed1999

Update of AMmtDB: a database of multi-aligned metazoa mitochondrial DNA sequences.

The present paper describes AMmtDB, a database collecting the multi-aligned sequences of vertebrate mitochondrial genes coding for proteins and tRNAs, as well as the multiple alignment of the mammalian mtDNA main regulatory region (D-loop) sequences. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. As far as the genes coding for tRNAs are concerned, the multi-alignments based on the primary and the secondary structures are both provided; for the mammalian D-loop multi-alignments we report the conserved regions of the entire D-loop (CSB1, CSB2, CSB3, the central region, ETAS1 and ETAS2) as defined by Sbisà et al. [ Gene (1997), 205, 125-140). A flatfile format for AMmtDB has been designed allowing its implementation in SRS (http://bio-www.ba.cnr.it:8000/BioWWW/#AMMTDB ). Data selected through SRS can be managed using GeneDoc or other programs for the management of multi-aligned data depending on the user's operative system. The multiple alignments have been produced with CLUSTALV and PILEUP programs and then carefully optimized manually.

Animals↗

PubMed1994

Internal translational initiation in the mRNA from the Neurospora crassa albino-3 gene.

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA.

Alkyl and Aryl Transferases↗

PubMed1998

A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1. 1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Amino Acid Sequence↗

PubMed1988

Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone.

One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3'-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.

Animals↗