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M Belfort

Publications and source records attributed to M Belfort.

At least 73 records · Page 4Linked to original sources
PubMed1992

The P2 element of the td intron is dispensable despite its normal role in splicing.

The P2 region of group I introns has been proposed to be involved in the correct positioning of the P1 5'-splice site duplex in the catalytic core (Michel, F., and Westhof, E. (1990) J. Mol. Biol. 216, 585-610). The behavior of delta P2 deletion mutants of the td intron is consistent with this hypothesis. The delta P2 mutants are capable of site-specific hydrolysis, indicating that the conformation of the ribozyme is not grossly altered, but they are incapable of transesterification reactions at the splice sites, as would be predicted if P1 is not appropriately aligned within the catalytic core. Nevertheless, the function of the P2 element can be bypassed in specific pseudorevertants isolated by genetic selection from the delta P2 mutants. These results, together with phylogenetic data, support the existence of alternate strategies to create a functional P1-core interaction.

Base Sequence↗
PubMed1992

Trans and cis requirements for intron mobility in a prokaryotic system.

Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient. The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process. To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds. Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively. The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda. Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance. We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination. Neither the precise position nor the nature of the double-strand break was critical to intron transfer. These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.

Adenosine Triphosphatases↗
PubMed1991

Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.

Fe(II)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higher-order folding of the group IB intron from Tetrahymena thermophila pre-rRNA. This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass. Significant portions of the phylogenetically conserved secondary structure are protected from Fe(II)-EDTA cleavage. However, the P4 secondary structure element, which is substantially protected in the Tetrahymena intron, is available for cleavage in the two T4 introns. We conclude that a family of catalytic RNAs (ribozymes) that possess similar secondary structures and have similar activities fold into similar but nonidentical tertiary structures that nevertheless serve to internalize portions of the catalytic center. Furthermore, comparison of cleavage patterns of the sunY and td intron RNAs indicates that conserved nucleotides outside as well as within the catalytic core participate in the tertiary structure.

Animals↗
PubMed1991

I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.

Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.

Base Sequence↗
PubMed1991

Effects of mutations of the bulged nucleotide in the conserved P7 pairing element of the phage T4 td intron on ribozyme function.

The P7 element of group I introns contains a semiconserved "bulged" nucleotide, a C in group IA introns (nt 870 in the td intron) and an A in group IB introns [Cech, T.R. (1988) Gene 73, 259-271]. Variants U870, G870, and A870, isolated by a combination of in vitro and in vivo genetic strategies, indicate that C and A at position 870 are consistent with splicing whereas U and G are not. Although mutants G870 and U870 could be activated in vitro by increasing the Mg2+ concentration, their Km for GTP at pH 7 was 20-100-fold elevated, and they were unable to undergo site-specific hydrolysis. The dependence of the mutants on high guanosine concentrations could be substantially overcome by an increase in pH, suggesting that a tautomeric change, which makes U and G mimic C and A, is responsible for restoring function. In contrast to the striking Km effect, Vmax for the mutants differed by less than a factor of 2 from the wild type. Furthermore, streptomycin, an aminoglycoside antibiotic that competes with guanosine for its binding site, inhibited splicing of the U870 and G870 constructs at least as well as of the C870 and A870 variants, indicating that the guanosine-binding site of the mutants is proficient at interacting with a guanidino group. While our experiments argue against a hydrogen-bonding interaction between the C6-O of the cofactor and C4-NH2 of the bulged nucleotide, they are consistent with other models in which the C4-NH2 and/or N3 groups of the bulged C are involved in establishing an active ribozyme.

Base Sequence↗
PubMed1991

Amniotic fluid glucose and intraamniotic infection.

Thirty-nine patients with either premature labor and/or preterm premature ruptured membranes underwent transabdominal amniocentesis to enable the following amniotic fluid analyses to be performed: culture and sensitivity, Gram's stain, and glucose determination. All nine patients with intraamniotic infection had amniotic fluid glucose values less than 10 mg/dl. Three patients with amniotic fluid glucose levels less than 10 mg/dl but without chorioamnionitis were delivered of infants within 72 hours of admission. The mean amniotic fluid glucose level of patients with intraamniotic infection (5 +/- 2.4 mg/dl) was significantly lower than in those without intraamniotic infection (39.8 +/- 18.42 mg/dl). All patients with amniotic fluid glucose values less than 10 mg/dl had either bacteria and/or white blood cells on Gram's stain. Two patients without chorioamnionitis had white cells on Gram's stain and amniotic fluid glucose values greater than 10 mg/dl. It appears that amniotic fluid glucose is more sensitive and more specific than Gram's stain in the diagnosis of intraamniotic infection. All 12 patients with low amniotic fluid glucose values were delivered of infants within 72 hours as the result of either the presence of infection or the progression of labor.

Amniotic Fluid↗
PubMed1991

Protein overproduction in Escherichia coli: RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane.

After optimizing overproduction of a heterologous gene product (chloramphenicol acetyltransferase, CAT) using an RNA stabilization vector * in Escherichia coli (Chan et al., 1988), a single step cell disruption and recovery method * for obtaining a product stream essentially free of cell debris was developed. The behavior of an RNA stabilization plasmid (pKTN-CAT) containing stabilizing intron RNA was investigated in two different media both in batch and chemostat modes. CAT production of pKTN-CAT was consistently higher (3- to 7-fold) than that of the control lacking the stabilization sequences (pK-CAT). Highest CAT production was observed for cells grown in minimal medium in batch mode and induced for CAT expression early in growth. CAT production of cells grown in the chemostat mode exhibited an optimal dilution rate of about 0.1 h-1. Enhancement of protein production by pKTN-CAT as compared to pK-CAT tended to be higher when grown in rich medium rather than in minimal medium. Presence of the RNA stabilization plasmid did not significantly alter the growth rate of the cell. Using a combination of chemical treatment (1 mM EDTA) and shear stress resulting from cross-flow in a stainless steel microfiltration membrane *, CAT was released into the medium through disruption of the E. coli cells. The permeate flux increased from 2000 to 9000 kg m-2 h-1 with increasing axial Reynolds number from 10,000 to 60,000 or increasing mean shear stress from 12 to 47 Pa. The turbidity of the permeate was approximately 4% that of the retentate over this range of axial flow rates, indicating excellent removal of cell debris. Also, the concentration of CAT in the permeate was equal to that in the retentate over this range of axial flow rates, indicating complete passage of protein through the membrane. Thus, using a combination of chemical treatment and fluid-induced shear stress in a cross-flow membrane module, we were able to disrupt and recover the heterologous protein in a stream low in debris.

Biotechnology↗
PubMed1991

A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion.

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

Alleles↗
PubMed1990

Spontaneous shuffling of domains between introns of phage T4.

The three self-splicing introns in phage T4 (in the td, sunY and nrdB genes) (Fig. 1a) each have the conserved group I catalytic RNA core structure (Fig. 1b), out of which is looped an open reading frame. Although the core sequences are very similar (approximately 60% identity), the open reading frames seem to be unrelated. Single crossover recombination events between homologous core sequences in the closely linked td and nrdB introns have led to 'exon shuffling. Here we describe spontaneous double crossovers between the unlinked td and sun Y introns that result in shuffling of an intron structure element, P7.1 (refs 3 and 4). The intron domain-switch variants were isolated as genetic suppressors of a splicing-defective P7.1 deletion in the td intron. This unprecedented example of suppression through inter-intron sequence substitution indicates that the introns are in a state of genetic flux and implies the functional interchangeability of the two analogous but nonidentical P7.1 elements. The implications of such recombination events are discussed in the light of the evolution of the introns themselves as well as that of their host genomes.

Base Sequence↗
PubMed1990

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications.

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5' and the sunY endonuclease 13-15 nt 3' to the intron insertion site to generate 3-nt or 2-nt 3'-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.

Amino Acid Sequence↗
PubMed1990

Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region.

Non-directed mutagenesis and phylogenetic comparison suggest that certain elements of the bacteriophage T4 td group Ia intron are dispensable to self-splicing. The L6-P6a-L6a region was identified as a potential non-essential element, and was removed by sequential deletions extending from the L6a loop toward the P6 pairing. Assays for splicing indicate that as long as the P6 pairing is maintained, the 1016 nucleotide td intron can be reduced to less than 250 nucleotides while maintaining function in vivo and in vitro. The P6 pairing appears to be essential for splicing while P6a is not. In addition, a spontaneous pseudorevertant of a splicing-defective deletion was isolated and shown to result from a single nucleotide change in the predicted L6a loop. This genetic suppressor mimics the ability of Mg2+ to reverse the phenotype of the deletion, suggesting that function is restored by structural stabilization of P6. The tolerance of this region to deletion prompted us to split the ribozyme core in L6a, to generate precursors that might function in trans. Indeed, the two half-molecules do associate to form a bimolecular complex that yields accurately ligated exons both in vitro and in vivo. The biological implications of these results, as well as the usefulness of trans-splicing for generating unprocessed precursors in vitro are discussed.

Base Sequence↗
PubMed1989

Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4.

Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6[5']) of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5'] and P6[3'] compensatory mutations. Second, all P6[5'] and P6[3'] mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5']:[3'] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.

Base Composition↗
PubMed1989

Mobile introns: definition of terms and recommended nomenclature.

A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases. Such introns are mobile genetic elements that insert themselves at or near the cleaved sites. A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.

Base Sequence↗
PubMed1989

A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4.

The product of the td intron open reading frame (ORF) of phage T4 is required for high-frequency transfer of the intervening sequence from intron-plus (In+) to intron-minus (In-) alleles. In vivo studies have demonstrated that the td ORF product targets cleavage of td In- DNA, and that cleavage is correlated with intron inheritance [Quirk et al., Cell 56 (1989) 455-465]. In the present study we show by in vitro synthesis of the td intron ORF product, that the protein possesses endonuclease activity and efficiently cleaves double-stranded DNA at or near the site of intron integration. In addition, we demonstrate that intron insertion is accompanied by co-conversion of the flanking exon sequences. Co-conversion of markers within 50 nt surrounding the site of intron insertion occurred at a high frequency (80-100%), and decreased at greater distance from the intervening sequence. Co-conversion may provide a mechanism for maintaining exon-intron RNA contacts required for accurate splicing of the relocated intron. Cleavage of target DNA by an intron endonuclease and co-conversion of flanking exon sequences are both features associated with mobile introns of eukaryotes, indicating a common mechanism for intron transfer in the eukaryotic and prokaryotic kingdoms.

Base Sequence↗
PubMed1989

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames.

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.

Cloning, Molecular↗