Analysis of bacteriophage Mu and λ-Mu hybrid DNAs by specific endonucleases

Allet, Bernard; Bukhari, Ahmad I.

doi:10.1016/0022-2836(75)90307-1

Cited by 167 publications

(57 citation statements)

References 8 publications

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“…Alu I generated 11 fragments (A to K in 6).nd the digestion products were used as marker DNAs, the chain lengths of which were taken from Allet and Bukhari (19) with Alu I fragments revealed that the one end of Alu Al was located at the 41st base pair from the labeled end of Hind II A (Fig. 3, also see Fig.…”

Section: Resultsmentioning

confidence: 99%

Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli

Sekiya¹,

Nishimura²

1979

Nucl Acids Res

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A DNA fragment of about 2000 base pairs carrying the gene for tRNAIle has been cloned from a total Eco RI endonuclease digest of Escherichia coli DNA. Sequence analyses revealed that about the first 850 base pairs from one end of the fragment contain a nucleotide sequence corresponding to that in the 3'-end of 16S rRNA. The gene for tRNAIle follows the 16S rRNA gene and both genes flank a spacer sequence of 68 base pairs. The spacer region contains a repeating, a hair pin and a symmetrical structure when the sequence is viewed in the single stranded form. A notable hair pin structure is also observed in the region adjacent to the 3'-end of the tRNAI e gene. In addition, about 850 base pairs from the other end of the DNA fragment have been found to contain the nucleotide sequence of the 5'-end of 23S rRNA. The presence of the genes for tRNAIle, 16S and 23S rRNA and the hybridization to tRNAl9a suggest that this cloned DNA is part of one of the E. coli rRNA operons carrying these two tRNA genes as a spacer.

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Section: Resultsmentioning

confidence: 99%

Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli

Sekiya¹,

Nishimura²

1979

Nucl Acids Res

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“…Bacteriophage Mu controls an unusual DNA-modification function (1,2) that protects viral DNA against cleavage by a variety of site-specific DNA endonucleases (3,4). The mom gene is located at the right end of the Mu genetic map in an operon comprising two overlapping genes.…”

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confidence: 99%

Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA.

Hattman

Newman

Murthy

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Bacteriophage Mu controls an unusual DNA-modification function encoded by the mom gene, which is located in an operon that consists of two overlapping genes. The com gene, located proximal to the 5' end of the common mRNA transcript, encodes a polypeptide of 62 amino acids that is required for translation of mom. Analysis of the derived amino acid sequence reveals that Com contains zinc-binding finger motifs, suggesting that Com may be a zinc-activated regulatory protein. Atomic absorption analysis showed that there is about one zinc bound per molecule of Com. We have subcloned the com gene into an expression vector and thus have overproduced and purified the Com protein. By gel retardation analysis with various 32P-labeled RNAs (made by in vitro transcription with T7 RNA polymerase), we show that Com binds specifically to com-mom mRNA. A single C----U substitution mutation, located 26 nucleotides upstream from the mom translation start codon, abolishes Com binding. The nature of the Com target sequence was deduced from in vitro footprinting analyses. The results are consistent with the existence of a complex stem-loop structure within the overlap of the com-mom open-reading-frames. Com binding to its target site results in the destabilization of a proposed translation-inhibitor stem-loop (TIS) to expose the Shine-Dalgarno sequence and mom translation initiation codon. This suggests that Com interaction with a specific site on its cognate mRNA alters the mRNA secondary structure to activate translation of mom.

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“…Bacteriophage Mu is known to control a DNA modification function (1) that protects its DNA against restriction by certain site-specific nucleases (2,3). The modification usually requires expression of both phage and host genes: (i) the phage Mu mom+ gene (1,4), which is located at the rightmost end of the genetic map; (ii) at least one other phage Mu (trans-acting) gene (5); and (iii) the host Escherichia coli dam' gene (6,7), which specifies a DNA-adenine methylase (8) that modifies the sequence G-A-T-C to G-m6A-T-C (9, 10).…”

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confidence: 99%

Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function.

Swinton¹,

Hattman²,

Crain³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Bacteriophage Mu encodes a protein that modifies "15% of DNA adenine residues to a new and unusual form. Modified DNA was enzymatically digested to deoxynucleosides, and the products were fractionated by HPLC. A modified adenine nucleoside, designated dA', was purified and its molecular structure was established by mass spectrometry. We show that dA' is a-N-9-4-D-2'-deoxyribofuranosylpurin--yl)-glycinamide. The dAx obtained from DNA was indistinguishable from the synthetic product with respect to its chromatographic behavior (HPLC and gas chromatography) and mass spectrum. Acid hydrolysis degrades dA' to produce N6-carboxymethyladenine; this compound corresponds to the base Ax observed in earlier studies.Bacteriophage Mu is known to control a DNA modification function (1) that protects its DNA against restriction by certain site-specific nucleases (2, 3). The modification usually requires expression of both phage and host genes: (i) the phage Mu mom+ gene (1, 4), which is located at the rightmost end of the genetic map; (ii) at least one other phage Mu (trans-acting) gene (5); and (iii) the host Escherichia coli dam' gene (6, 7), which specifies a DNA-adenine methylase (8) that modifies the sequence G-A-T-C to G-m6A-T-C (9, 10). It has recently been shown that the dam' methylase exerts a positive regulatory role in transcription of the phage Mu mom gene (11). Previously, the DNA modification was partially characterized, showing that ==15% of the adenine residues are modified (12) and that these residues are in specific sequences (3, 13). The modified base (designated Ax), observed in mild acid hydrolysates, was shown to contain a free carboxyl residue (12). The present communication reports the purification and molecular characterization of the modified deoxynucleoside. Mass spectrometric analysis identified the structure as a-N-(9-,B3D-2'-deoxyribofuranosylpurin-6-yl)glycinamide. Further investigation revealed that acid hydrolysis degrades this compound to produce N6-carboxymethyladenine; this base corresponds to the Ax observed in earlier studies (12). Ten 500-ml LB broth cultures of ND40 trp-. (Mucts62 lyslO25) were grown in 2-liter flasks at 32°C. At a titer of =3 X 108 cells per ml, the flasks were transferred to 42°C; after 30 min of incubation with shaking they were transferred to 37°C for 120 min. The cells were harvested by centrifugation and washed once in Ix NaCI/Cit, and the pellets were stored frozen at -20°C; the yield was 15 g (wet weight) of cells.The pellet was thawed and suspended in 150 ml of TEN buffer; 50 mg of crystalline egg white lysozyme was added, and the culture was gently swirled at 37°C. Lysis was completed by addition of 8 ml of 10% (wt/vol) Sarkosyl NL 97. Pronase (final concentration, 300 ,g/ml) was added and incubation continued for 3 hr at 37°C. Deproteinization was continued by extractions with phenol and then chloroform/isoamyl alcohol, 24:1 (vol/ vol). The aqueous phase was covered with 2 vol of 95% ethanol and the DNA was "spooled" on glass rods as described by Marm...

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Analysis of bacteriophage Mu and λ-Mu hybrid DNAs by specific endonucleases

Cited by 167 publications

References 8 publications

Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli

Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli

Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA.

Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function.

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Analysis of bacteriophage Mu and λ-Mu hybrid DNAs by specific endonucleases

Cited by 167 publications

References 8 publications

Sequence of the gene for isoleucine tRNA1and the surrounding region in a ribosomal RNA operon of Escherichia coli

Sequence of the gene for isoleucine tRNA1and the surrounding region in a ribosomal RNA operon of Escherichia coli

Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA.

Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function.

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Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli

Sequence of the gene for isoleucine tRNA₁and the surrounding region in a ribosomal RNA operon of Escherichia coli