A systematic DNA sequencing strategy

Hong, Guofan

doi:10.1016/0022-2836(82)90213-3

Cited by 201 publications

(71 citation statements)

References 19 publications

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“…The positive pools were subsequently reduced in size until the clone harbouring tzf-1 (pUSRT1) was isolated. In order to identify the position of t u f l on the BgllI insert of pUSRTl , DNaseI deletion derivatives were generated essentially as described by Hong (1982). The location of t#f-1 could be derived from the length of the insert, the Sall restriction pattern and Southern analysis which showed whether the Sall fragments that contain tuf sequences were still present.…”

Section: Methodsmentioning

confidence: 99%

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus

Vijgenboom¹,

Woudt²,

Heinstra

et al. 1994

Microbiology

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We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-7 gene encodes a protein with 71 O/ O amino acid residues identical to the well characterized elongation factor T u of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-7 downstream of a fus homologue and the in vitro activity of Sr.EF-Tul show that tuf-7 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EFTu. Homologues of tuf-7 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tul was present at all times under kirromycin production conditions in submerged and surface-grown cultures of 5. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tul was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of 5. ramocissimus.

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Section: Methodsmentioning

confidence: 99%

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus

Vijgenboom¹,

Woudt²,

Heinstra

et al. 1994

Microbiology

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“…T4 DNA ligase and restriction enzymes were purchased from Boehringer Mannheim Biochemicals and were used according to the manufacturer's specifications. Deletions were made with DNase I as described previously (12) or with exonuclease III as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype

et al. 1991

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We cloned and sequenced the sohB gene of Escherichia coli. The temperature-sensitive phenotype of bacteria that carry a Tn10 insertion in the htrA (degP) gene is relieved when the sohB gene is present in the cell on a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes a periplasmic protease required for bacterial viability only at high temperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on the E. coli chromosome, precisely between the topA and btuR genes. The gene encodes a 39,000-Mr precursor protein which is processed to a 37,000-Mr mature form. Sequencing of a DNA fragment containing the gene revealed an open reading frame which could encode a protein of Mr 39,474 with a predicted signal sequence cleavage site between amino acids 22 and 23. Cleavage at this site would reduce the size of the processed protein to 37,474 Mr. The predicted protein encoded by the open reading frame has homology with the inner membrane enzyme protease IV of E. coli, which digests cleaved signal peptides. Therefore, it is possible that the sohB gene encodes a previously undiscovered periplasmic protease in E. coli that, when overexpressed, can partially compensate for the missing HtrA protein function.

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“…There are approximately 20 heat shock genes that have been identified, all of which are under the positive control of the u32 transcription factor. This heat shock regulon includes genes whose products are proteases (e.g., Ion) and chaperonins, which assist the proper assembly and transport of proteins (e.g., groEL and dnaK), as well as the housekeeping transcription factor (0 itself (i.e., rpoD) (15,18,37).…”

mentioning

confidence: 99%

“…We conclude that the 35,000-Da protein is the htrB gene product and that the 10,000-Da protein could be a fortuitous open reading frame that may not be expressed extensively in vivo. In order to conclusively demonstrate that the inactivation of the 35,000-Da open reading frame is responsible for the HtrB phenotype, we constructed DNA deletions of plasmid pKS12 by using the DNase I method (20). The analysis of these deletions shows that there is a correlation between the presence of the 35,000-Da protein and the suppression of the HtrB phenotypes.…”

mentioning

confidence: 99%

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

et al. 1991

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We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33°C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB + gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates.

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A systematic DNA sequencing strategy

Cited by 201 publications

References 19 publications

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

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