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PubMed1987

Cloning and characterization of the aldA gene of Aspergillus nidulans.

We have cloned and sequenced the aldA (encoding aldehyde dehydrogenase) gene of Aspergillus nidulans. The gene contains two introns which are similar in size and structure to other fungal introns. The amino acid sequence of aldehyde dehydrogenase (497 residues) shows a significant level of homology with analogous sequences in other organisms. Comparison of the primary structure of the active sites of the mammalian cytosolic and mitochondrial enzymes shows that the Aspergillus enzyme closely resembles the mammalian mitochondrial enzyme. Analysis of the 5' non-coding region of the aldA gene shows a TATA-like sequence located 90 bp upstream from the initiation codon. Two messenger-RNA start points are located 36 and 42 bp upstream from the start codon.

Aldehyde Dehydrogenase↗

PubMed1994

Cloning and characterization of the gene encoding the major sigma factor of Stigmatella aurantiaca.

The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.

Amino Acid Sequence↗

PubMed2001

A reassessment of the translation initiation codon in vertebrates.

More than two decades ago Marilyn Kozak proposed the scanning model of translation initiation, whereby translation is initiated at the first AUG codon that is in a particular context. In this article, we re-examine the context of initiator codons using a large dataset of curated human transcripts. We find that more than 40% of transcripts contain AUG codons upstream of the actual start codon and that most authentic AUGs contain three or more mismatches from the consensus sequence, CCACCaugG. Also, in a large fraction of transcripts, the sequences surrounding the initiator codon deviate more from the consensus than those surrounding upstream AUGs, indicating that translation initiation from downstream AUGs is more common than generally believed.

5' Untranslated Regions↗

PubMed1999

Structural organization of the human gamma-glutamyl hydrolase gene.

Gamma-glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24 kb in the human genome, with nine exons sized from 51 to 371 bp. All of exon-intron splice junctions follow the GT-AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5' region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide -125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T-->C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.

Base Sequence↗

PubMed2001

Absence of effect of varying Thr-Leu codon pairs on protein synthesis in a T7 system.

The over-represented threonine-leucine (Thr-Leu) codon pair ACG CUG has been previously reported to be inhibitory to translation compared to the synonymous under-represented Thr-Leu codon pair ACC CUG, in an E. coli system in which the codon pairs were located either 3 and 4, or 6 and 7, or 9 and 10 codons downstream from the initiating codon for the message [Irwin, B., Heck, J. D., and Hatfield, G. W. (1995) J. Biol. Chem. 270, 22801-22806]. In the work reported here, these synonymous codon pairs were tested in a T7 system, with the codon pairs located either 14 and 15, or 6 and 7 codons downstream from the AUG start codon. In contrast to the reported findings in the E. coli system, there was no difference found in translation between mRNAs containing the respective codon pairs in the T7 system. The reasons for the different findings remain unclear, but presumably are a consequence of differences between the E. coli and T7 systems used to assay gene expression. Nevertheless, as a result of this work, it appears that the effect of varying codon pairs reported in the E. coli system is not due to a difference in translational step times through the respective codon pairs, as previously proposed.

Bacteriophage T7↗

PubMed1998

Cloning and characterization of the mouse histone deacetylase-2 gene.

Histone deacetylase-2 (HDAC2) is a component of a complex that mediates transcriptional repression in mammalian cells. A mouse HDAC2 cDNA was used to identify several recombinant clones containing the entire mouse HDAC2 gene. The mouse HDAC2 gene spans over 36 kilobase pairs and is composed of 14 exons (ranging from 58 to 362 nucleotides in length) and 13 introns (ranging from 75 base pairs to 19 kilobase pairs in length). Primer extension analysis with total RNA from NIH3T3 cells revealed a major transcriptional start site at 221 base pairs 5' of the ATG translational start codon. Upstream of the transcriptional start site, no canonical TATA box was found, but binding sites for several known transcription factors were identified. Transient transfection studies with 5' deletion mutants localized the promoter to no more than 76 base pairs upstream from the major transcriptional start site. Fluorescence in situ hybridization mapped mouse HDAC2 to chromosomal location 10B1, which is in close proximity to the growth factor-inducible gene fisp-12. Information concerning the genomic organization and promoter of HDAC2 will be useful in studies of the regulation of histone deacetylase activities, which in turn are important in studies of the regulation of transcriptional repression in mammalian cells.

Animals↗

PubMed2000

Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

Bacterial Proteins↗

PubMed2005

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.

Amino Acids↗

PubMed1998

Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus.

Two non-allelic laccase genes (lcc1 and lcc2) in Agaricus bisporus have been mapped to the same cosmid clone and are close together, in tandem. The intergenic region consists of 1562 bp between the stop codon of lcc1 and the start codon of lcc2. Differences between the 5' non-coding regions of the two genes suggest the potential for their differential regulation. By employing competitive RT-PCR and specific primer pairs that discriminate between lcc1 and lcc2, it has been shown that the level of lcc2 mRNA is approximately 300 times higher than that of lcc1 mRNA in malt extract liquid cultures; in compost cultures lcc2 mRNA is almost 7000 times more abundant than lcc1 mRNA.

Agaricus↗

PubMed2000

SUT2, a putative sucrose sensor in sieve elements.

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

Amino Acid Sequence↗

PubMed1986

Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. This study focused on the virC locus, which affects the host range Agrobacterium species. virC mutants display an attenuated or avirulent phenotype on certain host plants, but remain fully virulent on other plant hosts. The nucleotide sequence revealed that the virC locus of pTiA6NC is an operon consisting of two open reading frames. These two open reading frames, designated virC1 and virC2, encode protein products of 25,713 and 22,710 daltons, respectively, which were visualized by polyacrylamide gel electrophoresis. Only two nucleotides separated the stop codon for virC1 from the start codon for virC2, indicating that these genes may be translationally coupled.

Arginine↗

PubMed1996

Three functions of bacteriophage P1 involved in cell lysis.

Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.

Amino Acid Sequence↗

PubMed2001

Vector design for optimal protein expression.

Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is essential for stability, as well as for translation efficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of the sequences [5'UTR, 3'UTR, and poly(A) signal] are suggested.

3' Untranslated Regions↗

PubMedDate not supplied

Sequence similarities between the genes encoding the S.NgoI and HaeII restriction/modification systems.

The DNA sequence encoding the S.NgoI restriction/modification (R/M) system was identified from a gene bank made from Neisseria gonorrhoeae strain WR302 by identifying recombinant plasmids that induced the reporter system in a methylase detection strain AP1-200-9 (Piekarowicz et al., 1991) and were resistant to digestion with NgoI. The DNA sequence was determined from one of these (pUCP30). M.NgoI is a protein of 315 aa with a predicted MW of 35296 Da and R.NgoI is a protein of 350 aa with a predicted MW of 40650 Da. The termination codon of M.NgoI overlapped the start codon of R.NgoI. The same strategy was used to clone the R/M system encoding HaeII from Haemophilus aegyptius strain ATCC 11116. The DNA sequence from one clone representing this class (pAP704) was determined. HaeII methylase is a protein of 318 aa with a predicted MW of 35669 Da and R.HaeII contains 352 aa with a predicted MW of 40800 Da. aa alignments between the two methylases indicated that they were 74.3% identical and 79% similar. DNA sequence alignments revealed 68% identity. An aa alignment between the two restriction enzymes indicated that they were 60% identical and 68% similar. DNA sequence alignments revealed 61% identity. The DNA sequences flanking these two systems were identified and used to determine the genomic organization of the two systems. The S.NgoI genes were found between two genes, one with high homology to GTP binding proteins of unknown function and one with homology to genes involved in tRNA synthetase synthesis. The HaeII R/M genes were located between two genes, mucF and mucE. The DNA sequence of the HaeII R/M system was compared to the genomic DNA sequence of H. influenzae Rd. Although the DNA sequences flanking the HaeII system were > 99% identical to contiguous DNA fragments found in the genome of H. influenzae Rd, no homology was seen with the DNA sequences encoding the HaeII R/M system, indicating that it is not found in this strain. Given the vast difference in the GC content of S.NgoI and HaeII, their apparent insertion into polycistronic operons, and their difference in codon usage when compared to the species from which they were isolated, the data suggest that these R/M systems originated in an organism other than Neisseria or Haemophilus.

Amino Acid Sequence↗

PubMed1989

Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit.

Genetic reversion of HIS4 initiator codon mutations in yeast has identified three unlinked genes, sui1, sui2, and SUI3 (suppressors of initiator codon mutations), which when mutated confer the ability to initiate translation at HIS4 despite the absence of an AUG start codon. We have previously demonstrated that the SUI3 gene encodes the beta subunit of the eukaryotic initiation factor 2 (eIF-2) and that mutations at a Zn(II) finger motif of SUI3 alter the start site selection process in yeast. In this report, molecular and biochemical characterizations show that the sui2 suppressor gene encodes the alpha subunit of eIF-2. The amino acid sequence of sui2 is 58% homologous to that encoded by the cDNA of the human eIF-2 alpha. Mutations in the sui2 suppressor alleles occur in the amino-terminal portion of the protein and change amino acids that are identical at the same relative position in the yeast and human proteins. Protein sequence analysis shows that a sui2 mutant yeast strain allows initiation at a UUG codon in the absence of an AUG codon at HIS4. These data further suggest that eIF-2 is an important component of the preinitiation complex that mediates ribosomal recognition of a start codon during the scanning process.

Amino Acid Sequence↗

PubMed1988

Structures of the promoter and operator of the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12.

The nucleotide sequence of a 690-base-pair DNA segment containing the control region for the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase was determined. An ATG translation initiation codon with an adjacent ribosome-binding site was found which preceded an open reading frame continuing 61 codons to the end of the DNA that was sequenced. The start site for transcription, identified by using primer extension analysis, was located 42 base pairs upstream from the proposed Met start codon. The transcription start site was preceded by a region containing typical -10 and -35 sequences found in bacterial promoters. A binding site for the cyclic AMP-cyclic AMP receptor protein complex (identified by comparison with the consensus-binding sequence and verified by using DNase I footprinting) was located just upstream from the -35 sequence, centered at position -63. The interaction site for the glp repressor was identified by using DNase I footprinting. It consisted of a 49-base-pair region which started at the -10 sequence and continued to position +38. This region contained two directly repeated sequences, each possessing hyphenated dyad symmetry, which suggests that the operator is tandemly repeated. The presence of two adjacent operators may explain why expression of the glpD gene is the most sensitive to repressor when compared with expression of the other operons that are members of the glp regulon.

Aerobiosis↗

PubMed1986

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.

Amino Acid Sequence↗

PubMed1996

Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response.

This paper reports the expression of a previously described gene [Nath and Laal, Nucleic Acids Res. 18 (1990) 4935], currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system. The produced protein moved as a 95-kDa band on SDS-PAGE. An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence. A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon. The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding. Of functional significance was its immunoreactivity in human subjects with mycobacterial infection. Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

Adenosine Triphosphate↗