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PubMed2004

A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae.

The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.

Bacterial Adhesion↗

PubMed2004

Short-range compositional correlation in the yeast genome depends on transcriptional orientation.

This article reports an analysis of composition of about 5000 intergenic regions and neighboring ORFs in the nuclear genome of Saccharomyces cerevisiae, and their correlation. Intergenic regions flanked by divergently transcribed ORFs are GC richer (36%) than those separating convergent ORFs (29%). This difference in GC content cannot be fully attributed to its location upstream or downstream the ORFs, since no such strong compositional bias is found within 3' and 5' segments of intergenic regions between ORFs transcribed in the same direction. We have also found that the GC content of intergenic regions is positively correlated to that of its flanking ORFs in tandem and divergent orientations, but not in convergent orientations, and that the correlation coefficient between the GC content of nearby ORFs is higher for divergent pairs. Our observations are discussed in the light of recent work stressing the relationships between base composition, chromatin structure and meiotic recombination.

Base Composition↗

PubMed1987

The nucleotide sequence and genome organization of bovine papillomavirus type 4.

The nucleotide sequence of bovine papillomavirus type 4 (BPV-4) was determined. The viral genome is 7261 base pairs long. Several overlapping open reading frames (ORFs) have been identified both on the basis of amino acid comparison with other papillomaviruses and on their transcriptional pattern. Eight early ORFs (E1 to 8) were recognized, coding for DNA replication and cell transformation functions, and three late ORFs (L1 to 3), coding for structural proteins. Like the E5 ORF of human papillomavirus type 6 the E5 ORF of BPV-4 is discontinuous. Unlike other papillomaviruses, the non-coding region upstream of the early ORFs (ncr-1) is short (385 base pairs), but there is another non-coding region (ncr-2) of nearly 500 base pairs between the L2 and L1 ORFs. Most of the putative regulatory sites are located in the ncr-1, although potential controlling elements are also found in other parts of the genome. Polyadenylation sites are present at the 3' end of both the early and the late transcription units. Comparison between the polypeptides of BPV-4 and other papillomaviruses showed that BPV-4 is evolutionarily closer to the epitheliotropic human and rabbit viruses than to BPV-1.

Animals↗

PubMed1995

Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum.

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗

PubMed2000

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae.

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

Alleles↗

PubMed1997

Baculovirus gp64 gene expression: negative regulation by a minicistron.

Small upstream open reading frames (ORFs) or minicistrons located in the 5' leader of eukaryotic mRNAs have been shown to play a role in translational regulation of some eukaryotic genes, particularly mammalian proto-oncogenes. A survey of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus genome suggests that at least 10 transcripts from late genes contain potential minicistrons, and at least three of these minicistrons appear to be conserved in homologous genes of the related Orygia pseudotsugata MNPV. The position of the minicistron from one of these genes, gp64, is also conserved in gp64 genes from several baculoviruses, suggesting a potential regulatory function. To identify the potential role of the gp64 minicistron in regulating translation from gp64 late mRNAs, we generated a series of recombinant viruses containing the gp64 promoter and minicistron in combination with a chloramphenicol acetyltransferase reporter gene (cat) inserted into the polyhedrin locus. We first fused a cat reporter in frame with the minicistron coding region to demonstrate that the minicistron initiator ATG was in a context suitable for translational initiation. In subsequent experiments, a cat reporter was fused in frame to the downstream gp64 ORF, and various constructs containing point mutations that inactivated the minicistron were examined. Translational efficiency in the presence and absence of the minicistron was measured by quantitative analysis of gp64-cat RNA and the GP64-CAT protein. In the absence of a functional minicistron, translational efficiency from the downstream gp64-cat reporter ORF increased. Surprisingly, single-point mutations that inactivated the minicistron initiator ATG also resulted in utilization of an upstream in-frame ATG that is found within the minicistron coding region and that is in a poor translational initiation context. Double-point mutation constructs that inactivated both the minicistron initiator ATG and the upstream in-frame ATG also resulted in increased translational efficiency from the downstream gp64-cat ORF. Thus, the gp64 minicistron serves as a negative regulatory element that decreases translation of the gp64 ORF on late mRNAs.

Animals↗

PubMed1987

Three class I introns in the ND4L/ND5 transcriptional unit of Neurospora crassa mitochondria.

The overlapping ND4L and ND5 genes of Neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. All three intervening sequences are class I introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. They contain long open reading frames (ORFs; from 306 to 425 codons long) that are continuous and in frame with the upstream exon sequences. These ORFs contain the conserved decapeptide-encoding sequences that are characteristic of the ORFs present in most class I introns. Extensive homology exists among the ORFs encoded by the ND4L intron, ND5 intron 1, and the second intron of the N. crassa oli2 gene. Also, internal repeats of about 130 amino acid residues are present twice in each of these three ORFs, suggesting that a duplication event may have occurred in the formation of these ORFs. The ND4L intron shares extensive homology (at the levels of both primary and proposed secondary structures) with the self-splicing intervening sequence present in the Tetrahymena nuclear rRNA gene. This homology includes but is not limited to the core secondary structure, as peripheral structural elements are also conserved in the two introns.

Amino Acid Sequence↗

PubMed2001

Location, nucleotide sequence, and regulation of the p51 late gene of the hz-1 insect virus: identification of a putative late regulatory element.

An Hz-1 insect virus (Hz-1V) late gene encoding, a predicted polypeptide of 51 kDa was isolated from a cDNA library and mapped to the HindIII-J region (40-44.6 map units) of the viral genome. The p51 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 1,152 bp open reading frame (ORF) is transcribed as a 1.8 kb RNA between 8 and 18 h post-infection (hpi) with maximum expression at 12 hpi. Homology was not detected between the nucleotide sequence upstream of the p51 ORF and the baculovirus conserved late promoter element NTAAG. Primer extension analysis detected one major late transcription initiation site at -205 nucleotides relative to the start of the p51 ORF and seven minor late initiation sites at positions upstream of this primary site. Comparison of the upstream regulatory regions of the p51 gene and the Hz- 1V p34 late gene revealed a region of significant homology comprised of the 9 bp sequence TTATAGTAT. The primary p51 transcription initiation site and all p34 transcription initiation sites were mapped to different nucleotides within this nonanucleotide sequence. This 9 bp motif was not observed in the ORFs of these genes, and no significant homology was detected between this motif and the 5' regulatory regions of any other characterized genes. The results of our study suggest that this conserved sequence may serve an important role in the regulation of Hz-1V late genes.

Amino Acid Sequence↗

PubMed1988

Sequence analysis of the wheat mitochondrial atp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins.

The nucleotide sequence of the wheat mitochondrial gene for subunit 6 (atp6) of the F1F0 ATPase complex has been determined. Unlike bacterial, chloroplast or animal/fungal mitochondrial atp6 counterparts, which encode proteins of about 230-270 amino acids, the wheat mitochondrial atp6 homologue comprises the latter part of an open reading frame (ORF) of 386 codons. The ATP6 protein may therefore by synthesized with a long N-terminal presequence. This is supported by the finding that the ORF is preceded by a conserved sequence block closely related to ones preceding several other actively transcribed wheat mitochondrial protein-coding genes. The fused upstream ORF is similar in length, but unrelated in sequence, to those preceding the maize and tobacco mitochondrial atp6 genes. In wheat, the atp6 gene is located on a recombinationally active repeated DNA element, whose length of 1.4 kb corresponds approximately to that of the atp6 mRNA. A comparison of the wheat and maize ATP6 sequences reveals unexpectedly high divergence in the region corresponding to the mature N-terminal domain and may reflect mitochondrial DNA rearrangements during atp6 gene evolution in monocotyledonous plants.

Amino Acid Sequence↗

PubMed1996

Definition of the single integration site of the pathogenicity locus in Clostridium difficile.

We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc. Sequencing of the appropriate stretch of DNA in C. difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains. Our data define the locus as a distinct genetic element. Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined. This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains. Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself. Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence. The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein. A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed. Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C. difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.

ATP-Binding Cassette Transporters↗

PubMed1999

The catalytic group-I introns of the psbA gene of chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications.

The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1-Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3' end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11-38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas.

Algal Proteins↗

PubMed2003

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Actl7b, represses its expression in somatic cells.

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5'-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5' upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.

Actins↗

PubMed2000

Polymorphic open reading frames encoding secretory proteins are located less than 3 kilobases from Theileria parva telomeres.

Polymorphic, multicopy gene families are frequently located in subtelomeric regions of the genomes of parasitic protozoa. Theileria parva telomere-associated (TA) DNA from two chromosomes contained long open reading frames (ORFs) 54% identical at the N-termini, whose 3' ends were 2670 and 2680 bp from the telomeric repeats. Probes derived from these ORFs revealed related sequences close to additional telomeres. The 3' end of an unrelated ORF was approximately 2720 bp from a third telomere. These are among the closest ORFs to telomeres in any organism. Reverse transcription PCR detected transcripts originating within the telomeric multicopy gene family. Additional ORFs, with complex sequence similarities, were located centromeric to the telomere-adjacent ORFs. Transcripts from the schizont stage of T. parva, containing domains with significant amino acid similarity to a 3529 codon ORF located 6900 bp upstream of the telomeric repeats, were mapped to a subtelomeric locus at a fourth telomere. Five telomeric ORFs contained predicted N-terminal signal peptides and one of these signal peptides was functional in a heterologous system. Hybridisation data suggested extensive strain polymorphism between ORFs. Two of the telomere-adjacent ORFs were absent from the genome of a cloned T. parva parasite which can, nonetheless, be passaged through ticks and cattle. T. parva is unusual, among organisms so far studied, in the high density of potential coding sequences located directly adjacent to telomeres and the apparent absence of extensive tracts of repeated sequences within the TA DNA.

Amino Acid Sequence↗

PubMed1990

A lipopeptide-encoding sequence upstream from the lysA gene of Pseudomonas aeruginosa.

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.

Amino Acid Sequence↗

PubMed1990

Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems.

Streptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved in Escherichia coli by the construction of two-cistron expression systems formed in the high copy number plasmid vector pUC119. These two-cistron constructions were composed of the IPNS gene and its flanking sequences which encoded an upstream open reading frame (ORF), the IPNS ribosome binding site and a putative transcription terminator. No E. coli- like Streptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by the lac promoter of pUC119. Enzymatically active IPNS was detected in E. coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production of S. clavuligerus IPNS in E. coli. These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins in E. coli.

Base Sequence↗

PubMed1994

Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.

Amino Acid Sequence↗

PubMed1995

The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: regulatory elements and nucleosome positioning motifs.

We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3' end of each transcription unit. Located approximately 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; approximately 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.

Amino Acid Sequence↗

PubMed2000

Esr genes show different levels of expression in the same region of maize endosperm.

Esr genes share high homology among each other, code for small hydrophilic proteins, and are expressed in a restricted region of maize endosperm surrounding the embryo. We show here that not only Esr2 but also Esr1 and Esr3 are expressed in maize, and that the relative contribution of Esr1, Esr2 and Esr3 to total Esr mRNA is 17%, 55% and 28%, respectively. DNA sequence analysis of putative promoter fragments ranging from 0.53 kb to 3.54 kb revealed the presence of retrotransposons related to the Zeon and Cinful families in the distal parts of the promoters. The proximal parts show high homology that extended over 504bp between Esr2 and Esr3, and 265bp between Esr1 and the other two genes. The most conspicuous potential cis element is a fully conserved tandem repeat of the sequence CTACACCA close to the respective open reading frames (ORFs). By the analysis of transgenic maize plants carrying promoter-Gus fusions, it was shown that all three cloned upstream fragments contain functional promoters, that the spatial activity of all three Esr promoters is identical, and that the cis element(s) responsible for the expression in the embryo surrounding region reside in the 265 bp upstream of the respective ORFs.

Base Sequence↗