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M Belfort

Publications and source records attributed to M Belfort.

At least 91 records · Page 5Linked to original sources
PubMed1989

The inconsistent distribution of introns in the T-even phages indicates recent genetic exchanges.

Group I self-splicing introns are present in the td, nrdB and sunY genes of bacteriophage T4. We previously reported that whereas the td intron is present in T2, T4 and T6, the nrdB intron is present in T4 only. These studies, which argue in favor of introns as mobile genetic elements, have been extended by defining the distribution of all three T4 introns in a more comprehensive collection of T2, T4 and T6 isolates. The three major findings are as follows: First, all three introns are inconsistently distributed throughout the T-even phage family. Second, different T2 isolates have different intron complements, with T2H and T2L having no detectable introns. Third, the intron open reading frames are inherited or lost as a unit with their respective flanking intron core elements. Furthermore, exon sequences flanking sites where introns are inserted in the T4 td, sunY and nrdB genes were determined for all the different T-even isolates studied. Six of eighteen residues surrounding the junction sequences are identical. In contrast, a comprehensive comparison of exon sequences in intron plus and intron minus variants of the sunY gene indicate that sequence changes are concentrated around the site of intron occurrence. This apparent paradox may be resolved by hypothesizing that the recombination events responsible for intron acquisition or loss require a consensus sequence, while these same events result in sequence heterogeneity around the site.

Base Sequence↗
PubMed1989

Bacteriophage introns: parasites within parasites?

Several bacteriophage introns carry the information necessary to perform a complex series of RNA as well as DNA rearrangements. The RNA splicing reactions allow expression of the intron-containing genes, whereas recombination at the DNA level results in the mobility and invasive potential of the introns. The RNA and DNA transactions are temporally regulated to permit timely splicing early in infection, and expression of intron-encoded DNA endonucleases involved in mobility at late times. This is achieved through efficient use of the viral regulatory machinery, ensuring the perpetuation of the 'parasitic' introns.

Animals↗
PubMed1989

Haemodynamic changes in gestational proteinuric hypertension: the effects of rapid volume expansion and vasodilator therapy.

Ten patients with gestational proteinuric hypertension were studied with a Swan-Ganz thermodilution haemodynamic catheter before, during and after plasma volume expansion. Five patients were treated with dihydralazine before volume expansion and five after volume expansion. Before treatment all patients had a low pulmonary capillary wedge pressure (PCWP), low cardiac index (CI) and high systemic vascular resistance (SVR). Following volume expansion the PCWP and CI increased, the SVR decreased but the blood pressure (BP) was unchanged. Administration of dihydralazine following volume expansion led to a decrease in PCWP, an increase in CI and a decrease in SVR and BP. Dihydralazine alone caused an increase in heart rate, PCWP, and CI, and a decrease in SVR and BP. Volume expansion, by increasing CI and decreasing SVR, may be of therapeutic benefit in the severely hypertensive pregnant patient with a low cardiac index.

Adult↗
PubMed1988

Stability of group I intron RNA in Escherichia coli and its potential application in a novel expression vector.

Intron RNA excised from the primary transcript of the phage T4 td gene was found to be unusually stable in vivo. In contrast to the average half-life of about 1.5 min for a typical Escherichia coli mRNA at 37 degrees C, the half-life of the excised group-I td intron ranged from 12 to 19 min for the linear form and from 22 to 33 min for the circular form. A 631-nucleotide region of the intron that is not essential for splicing was replaced by the chloramphenicol acetyltransferase (CAT) structural gene (cat). Although the presence of the foreign sequence reduced intron stability several-fold, the half-life of the resulting intron-cat hybrid RNA was found to be twice that of the normal cat mRNA. The increase in stability was accompanied by a five- to eight-fold increase in CAT production above that seen with transcriptional activation from the strong Ptac promoter alone. The over-production was both temperature-dependent and partially splicing-dependent. This type of intron fusion represents a novel method of transcript stabilization, which is of potential use to augment other means of increasing gene expression for purposes of product amplification.

Chloramphenicol O-Acetyltransferase↗
PubMed1988

Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes.

Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the three introns. All three introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.

Animals↗
PubMed1988

Genes within genes: independent expression of phage T4 intron open reading frames and the genes in which they reside.

The td, nrdB, and sunY introns of bacteriophage T4 each contain a long open reading frame (ORF). These ORFs are preceded by functional T4 late promoters and, in the case of the nrdB intron ORF, a functional middle promoter. Expression of phage-encoded intron ORF-lacZ fusions indicates that these T4 genes are highly regulated. The lack of translation of these ORFs from early pre-mRNAs can be accounted for by the presence of secondary structures that are absent from the late RNAs. Because translation of the intron ORFs could disrupt core structural elements required for pre-mRNA splicing, such regulation may be necessary to allow expression of the genes in which they reside.

Cloning, Molecular↗
PubMed1987

Structural conventions for group I introns.

Conventions for nomenclature of structural elements and a standard secondary structure representation for group I introns have been established by workers in the field. These conventions are designed to facilitate effective communication of information concerning the structure and function of these self-splicing introns.

Animals↗
PubMed1987

Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility.

The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence. The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6. Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies. The results indicate the nonessential nature of the intron in nrdB expression and phage viability. Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor. Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.

DNA Transposable Elements↗
PubMed1987

Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis.

Of 97 nondirected T4 thymidylate synthase-defective (td) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5' domain and tdN47 destabilizing the P9 helix in the 3' domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4tdN57 and T4tdN47, both of which are impaired in cleavage at the 5' and 3' splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.

Base Sequence↗
PubMed1987

Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant.

A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.

Base Sequence↗
PubMed1986

Cloning and expression of an intron-deleted phage T4 td gene.

The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis. Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleotides from the 3'-end (pKTdX-1), or a deletion of two-thirds of the central portion (pKTd delta 1-3). Slightly more synthase accumulated in cells carrying pKTd delta I as compared to the other td genes when induction was performed at 30, 37, or 42 degrees C. Dramatically different results were observed in vitro, where enzyme activity synthesized from pKTd delta I DNA appeared earlier and reached severalfold higher levels than with pKTd2 DNA. In addition, thymidylate synthase expression from pKTdX-1 was impaired relative to pKTd2, while pKTd delta 1-3 accumulated enzyme at levels intermediate between those of pKTd2 and pKTd delta I. Under both in vivo and in vitro conditions, increasing levels of mature td mRNA preceded and paralleled those in enzyme activity for all four plasmids, demonstrating comparable translation of the mRNAs produced. From these results it would appear that the splicing of td RNA is much more efficient in vivo than in vitro, suggesting that other cellular components may facilitate in vivo processing of this intron-containing transcript.

Chromosome Deletion↗
PubMed1986

Multiple self-splicing introns in bacteriophage T4: evidence from autocatalytic GTP labeling of RNA in vitro.

RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions. One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe. These mapped in or near the unlinked genes nrdB and nrdC. A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro. Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4.

Genes, Viral↗
PubMed1986

Characterization of the intron in the phage T4 thymidylate synthase gene and evidence for its self-excision from the primary transcript.

The td gene contains a 735 bp open reading frame within its 1017 bp intron. A 12 nucleotide stretch may form a stable secondary structure with the putative Shine-Dalgarno sequence of the intron open reading frame and thus impair its translation. SP6 RNA polymerase transcripts of the td gene synthesized in vitro at 40 degrees C encompass a 2.7 kb primary transcript, a 1.7 kb mRNA, and a 1 kb intron RNA. The excised intron RNA consisted of linear and cyclized forms. RNAase H studies and resistance of the cyclized intron to linearization by HeLa cell debranching enzyme suggest it to be circular. Self-splicing of isolated td primary transcript occurred only marginally at 28 degrees C, but increased progressively to 50 degrees C, and required the presence of both Mg++ and a guanosine cofactor. An internal guide sequence is evident which may align the 5' splice site with the 3' end, presumably for precise exon ligation.

Base Sequence↗
PubMed1986

RNA splicing and in vivo expression of the intron-containing td gene of bacteriophage T4.

The splice junction sequence of td mRNA from T4-infected cells has been determined (5'....GGU-CUA....3') and shown to be identical to that of the RNA ligation product encoded by the cloned gene [Belfort et al. Cell 41 (1985) 375-382]. The RNA processing functions, T4 RNA ligase, T4 polynucleotide kinase, and the host prr gene product appear not to be essential for exon ligation; neither are the host endoribonucleases RNase III, RNase P and RNase E required for intron excision. While these results are consistent with the autocatalytic splicing mechanism demonstrated in vitro [Chu et al. J. Biol. Chem. 260 (1985) 10680-10688], they leave unanswered the question of which protein(s), if any, might stimulate the in vivo reaction. Analysis of the products of the cloned td gene has led to identification of two td-encoded polypeptides, namely a polypeptide corresponding to the exon-I-coding sequence (NH2-TS), and the catalytically active thymidylate synthase (TS). Kinetic and nucleotide sequence data provide evidence that NH2-TS is the product of the primary transcript and that TS is encoded by spliced mRNA. These results suggest that splicing may provide a switch controlling the relative expression of NH2-TS and TS, two proteins with markedly different temporal appearances despite their identical transcriptional and translational start sites.

Base Sequence↗
PubMed1986

Processing of phage T4 td-encoded RNA is analogous to the eukaryotic group I splicing pathway.

Several features of the split td gene of phage T4 suggest an RNA processing mechanism analogous to that of the self-splicing rRNA of Tetrahymena and other group I eukaryotic introns. Previous work has revealed conserved sequence elements and the ability of td-encoded RNA to self-splice in vitro. We show here that a noncoded guanosine residue is covalently joined to the 5' end of the intron during processing. Further, we demonstrate the existence of linear and circular intron forms in RNA extracted from T4-infected cells and from uninfected Escherichia coli expressing the cloned td gene. Sequence analysis of the intron cyclization junction indicates that the noncoded guanosine and one additional nucleotide are lost from the 5' end of the intron upon cyclization. This analysis places a uridine residue upstream of the cyclization site, in analogy to three other group I cyclization junctions. These striking similarities to the splicing intermediates of eukaryotic group I introns point not only to an analogous processing pathway and conserved features of cyclization site recognition but also to a common ancestry between this prokaryotic intervening sequence and the group I eukaryotic introns.

Animals↗
PubMed1985

In vitro expression of the intron-containing gene for T4 phage thymidylate synthase.

The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template. The [3H]leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000. The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene. When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained. Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II. Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies. The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide. The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed. Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.

Base Sequence↗