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D A Shub

Publications and source records attributed to D A Shub.

At least 37 records · Page 2Linked to original sources
PubMed1991

The product of the split sunY gene of bacteriophage T4 is a processed protein.

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.

Genes, Viral↗
PubMed1990

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria.

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.

Animals↗
PubMed1990

A self-splicing group I intron in the DNA polymerase gene of Bacillus subtilis bacteriophage SPO1.

We report a self-splicing intron in bacteriophage SPO1, whose host is the gram-positive Bacillus subtilis. The intron contains all the conserved features of primary sequence and secondary structure previously described for the group IA introns of eukaryotic organelles and the gram-negative bacteriophage T4. The SPO1 intron contains an open reading frame of 522 nucleotides. As in the T4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in a highly conserved region of the intron core. The exons encode SPO1 DNA polymerase, which is highly similar to E. coli DNA polymerase I. The demonstration of self-splicing introns in viruses of both gram-positive and gram-negative eubacteria lends further evidence for their early origin in evolution.

Amino Acid Sequence↗
PubMed1990

Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns.

The catalytic core of the sunY intron of bacteriophage T4 is separated from its 3' exon by 837 nucleotides, most of which are part of an open reading frame (ORF). Here, we report that transcripts truncated within the sunY ORF self-splice in vitro to a variety of sites in the segment immediately 3' of the core. Recognition of these proximal splice sites is shown to depend on (1) the presence on the intron side of a terminal G, which must not be part of a secondary structure; and (2) the ability of the penultimate intron nucleotide to base-pair with a 3' splice site-binding sequence (3'SSBS) located within the core. The counterpart of the 3'SSBS can be identified in most group I introns. The possible significance of such alternative splicing events for in vivo expression of intron-encoded proteins is discussed.

Base Composition↗
PubMed1989

The catalytic core of the sunY intron of bacteriophage T4.

The self-splicing sunY intron of bacteriophage T4 shares a common secondary structure with other group I introns. A long open reading frame within the intron is entirely 3' of the structural elements conserved in all group I introns. This catalytic core is the smallest yet described for a self-splicing intron. An internal deletion of 728 nucleotides (nt), leaving 196 nt at the 5' end and 109 nt at the 3' end, allows normal self-splicing. Transcripts terminating 196 nt 3' of the 5' splice site retain catalytic activity.

Catalysis↗
PubMed1989

Exon shuffling by recombination between self-splicing introns of bacteriophage T4.

The organization of genes into exons separated by introns may permit rapid evolution of protein-coding sequences by exon shuffling. Introns could provide non-coding targets for recombination, which would then give rise to novel combinations of exons. Evidence to support this theory is indirect and consists of examples of homologous domains of protein structure encoded in different genes, with introns in conserved positions at the boundaries of these domains. Here, we report the first direct evidence for exon shuffling. Two spontaneous deletion mutations of phage T4 have been characterized by sequencing, and they are clearly the result of recombination between homologous regions of two self-splicing group I introns. As a result of the recombination, exons of different genes are transcribed together, with a hybrid intron between them. One of these introns is proficient in self-splicing.

Exons↗
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↗
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↗
PubMed1985

Bacteriophage T4, a new vector for the expression of cloned genes.

The amino-terminal portion of the T4 rIIB gene has been fused to the coding sequence of a truncated lacZ gene from Escherichia coli, giving rise to a fusion protein with beta-galactosidase activity. The 3192-bp rIIB-lacZ gene fusion was transferred into phage T4, and enzymatically active protein was produced after phage infection. T4 may be a useful expression vector in special circumstances, in particular for proteins whose accumulation in E. coli is limited by sensitivity to proteases.

Cloning, Molecular↗
PubMed1985

Mapping the genes in the terminal redundancy of bacteriophage SPO1 with restriction endonucleases.

Although most early transcription from SPO1, a lytic DNA bacteriophage of Bacillus subtilis, is specified by the 12.6-kilobase region of the terminal redundancy, early genes from this region have not been identified by standard genetic means. We mapped genes to DNA regions of the SPO1 terminal redundancy by analyzing in vitro protein synthesis from isolated SPO1 restriction fragments in an Escherichia coli-coupled transcription-translation cell-free system. DNA from the terminal redundancy directs the synthesis in vitro of eleven proteins, e3, e4, e6, e7, e9, e12, e15, e16, e18, e20, and e21, which correspond in mobility on sodium dodecyl sulfate-polyacrylamide gels with authentic SPO1 early proteins. From their mapped positions on the DNA, genes were positioned downstream from most, but not all, of the twelve early promoter regions identified in vitro in the terminal redundancy. The temporal patterns of early protein synthesis in vivo suggest a differential turning on and off of early promoters in the terminal redundancy. Both in vivo and in vitro evidence suggests the existence of previously unidentified early promoter regions upstream from the genes for e6 and e4 as well as a middle promoter region upstream from the gene for e16.

Bacillus subtilis↗
PubMed1982

Bacteriophage T4 as a generalized DNA-cloning vehicle.

We have designed a method for inserting foreign DNA segments into bacteriophage T4. A plasmid containing T4 DNA is opened within the T4 sequence and the foreign DNA is inserted in vitro. Recombination in vivo, between T4 and the doubly chimeric plasmid, results in insertion of the foreign DNA into the genome of viable T4 phage. We have demonstrated the method by inserting a 203-bp DNA fragment from the lactose operon of Escherichia coli, into the dispensable region of the rIIB gene of T4. With minor modifications, the method should make possible the cloning of very large DNAs into any one of a large number of sites on the T4 chromosome.

Cloning, Molecular↗
PubMed1982

Transcriptional regulation of bacteriophage SPO1 protein synthesis in vivo and in vitro.

There are six classes of SPO1 transcripts which are, at least partially, regulated independently of each other. Analysis of proteins made in infections by phage mutants defective in DNA synthesis, or in genes which positively control transcription, permitted each protein to be assigned to one transcription class. Most of the late proteins belong to transcription class m2l. There seem to be few, if any, phage proteins in the l class whose mRNA synthesis depends absolutely on phage DNA synthesis, UV irradiation of host cells allowed the detection of many additional early proteins. The early proteins detected in vivo were compared with proteins synthesized in vitro, using bacterial or gp28 phage-modified RNA polymerase in an Escherichia coli cell-free system. Proteins characterized in vivo as belonging to the e transcription class could be made efficiently in vitro only when transcription was performed by bacterial RNA polymerase. em proteins could be elicited through the use of either bacterial or gp28 polymerase, indicating that their genes can be transcribed in either the early or the middle mode.

Bacillus subtilis↗
PubMed1979

The nature of transcription selectivity of bacteriophage SPO1-modified RNA polymerase.

Escherichia coli and Bacillus subtilis RNA polymerase have almost identical transcription specificities on bacteriophage SPO1 DNA when assayed in a coupled transcription-translation cell free system. SPO1-modified B. subtilis RNA polymerase has altered transcription specificity. It is shown that rifampicin-inhibited E. coli RNA polymerase can completely block transcription of SPO1 DNA by rifampicin resistant B. subtilis enzyme, whereas it has no effect on transcription by SPO1-modified B. subtilis RNA polymerase. We conclude that the new transcription by SPO1-modified RNA polymerase results from newly recognized promoters, rather than by elongation of transcripts which could also be made by B. subtilis vegetative RNA polymerase.

Bacillus subtilis↗
PubMed1975

Bacteriophage SPO1 DNA- and RNA-directed protein synthesis in vitro: the effect of TF1, a template-selective transcription inhibitor.

Transcription factor one (TF1), a protein synthesized after infection of B. subtilis by phage SPO1, is a specific inhibitor of SPO1 in vitro transcription. In this paper, we investigate the effect of TF1 on SPO1-specific in vitro protein synthesis, using SPO1 DNA or messenger RNA as templates. Protein syhthesis is measured by incorporation of radioactive amino acids into acid insoluble form, synthesis of a phage-specific enzymatic activity, and analysis of radioactive polypeptides by acrylamide gel electrophoresis. TF1 is shown to inhibit the DNA-dependent synthesis of all SPO1 proteins which can be made in vitro, but to have no specific effect on RNA-dependent protein synthesis in vitro.

Bacillus subtilis↗
PubMed1975

Bacteriophage SPO1 DNA- and RNA-directed protein synthesis in vitro: comparison with in vivo control.

A cell free protein synthesizing system, derived from E. coli, is shown to be a quantitiative assay system for messenger RNA extracted from B. subtilis infected with bacteriophage SPO1. DNA-directed protein synthesis in this system is shown to be limited mostly to those proteins whose messages are contained in early RNA. A phage induced enzyme, dCMP deaminase, is shown to be dependent on appearance of a class mRNA made in vivo in response to new initiations of transcription dependent on prior synthesis of phage induced protein. Control of the enzyme synthesized in the cell free system is contrasted with in vivo control, and an estimate of "read-through" by RNA polymerase in vitro is presented.

Bacillus subtilis↗