Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

Rood, Julian I.; Laird, Alan; Williams, Jeffrey W.

doi:10.1016/0378-1119(80)90003-7

Cited by 40 publications

(20 citation statements)

References 17 publications

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“…A summary of some properiies of the DHIFR from these transformed strains and from D3-157 and H712 is shown in Table 3. The inhibition by TMP and specific activities were consistent with those of previous transformants of wild-type DHFR strains containing these plasipids (11,13).…”

supporting

confidence: 88%

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

1986

J Bacteriol

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supporting

confidence: 88%

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

1986

J Bacteriol

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“…Substrate Proteins-Protease substrates were derived from barnase, a ribonuclease from Bacillus amyloliquefaciens (32), and dihydrofolate reductase (DHFR) 1 from Escherichia coli (33). The two proteins were linked in-frame, with barnase at the N terminus followed by DHFR.…”

Section: Methodsmentioning

confidence: 99%

Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

Lee

Prakash

Matouschek

2002

Journal of Biological Chemistry

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The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.Protein degradation is a critical part of cellular regulation (1). In eukaryotic cells, a multicomponent protease called the proteasome is responsible for the turnover of short-lived regulatory proteins, the removal of abnormal polypeptides, and the production of peptides for antigen presentation (2). Degradation of short-lived regulatory proteins is essential for a wide range of cellular functions including cell cycle control and signal transduction (3). Failure to degrade aggregates of misfolded proteins can lead to disease such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease (4). Degradation by the proteasome usually involves two consecutive steps: targeting of the substrate for degradation by the attachment of polyubiquitin chains and degradation of the tagged protein by the proteasome with concomitant release of ubiquitin (5).The active sites of proteolysis of the proteasome by themselves show little substrate specificity. Specificity of degradation is achieved by sequestering the proteolytic sites within the structure and tightly controlling access. The three-dimensional structure of the proteasome has been determined by x-ray crystallography and electron microscopy (6 -11). The proteasome consists of a central proteolytic core particle with regulatory caps at either end of it. The core particle is made of two copies of seven different ␣-subunits and seven different ␤-subunits. The subunits are arranged in four heptameric rings, which are stacked on top of each other to form a cylindrical particle. The rings form a central ␤-chamber and two ␣-chambers, one at each end of the particle (2). The proteolytic sites are located in the ␤-chamber and are accessible only through a central channel that runs along the long axis of the particle. The channel has narrow constrictions at the entrance and exit of the ␣-chamber (2). In the isolated yeast core particle, the entrance to the degradation channel is blocked by the N termini of the ␣-subunits (7). The channel opens when the regulatory caps bind to the core (12). The caps consist of 17 subunits, six of which have ATPase activity, and contact the co...

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“…Although several mechanisms of trimethoprim resistance in E. coli have been described (35), the most common mechanism is an increase in folA expression (3,11,28,36). To determine whether the trimethoprim resistance conferred by pAC1 was caused by expression of V. harveyi folA, we PCR amplified folA from BB7 and BB7X cells by using the primers 5Ј-CTATCGCCGTGG ATCCATAGTTTAA and 5Ј-TTGCGGTGCTTTCTGCAG TCTATTC and cloned the fragments onto the high-copy plasmids pCR2.1-TOPO and pUC18 and the low-copy plasmid pGD103.…”

Section: Vol 188 2006mentioning

confidence: 99%

TheVibrio harveyiGTPase CgtA_VIs Essential and Is Associated with the 50S Ribosomal Subunit

et al. 2006

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It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtA V is not essential. Here we show that cgtA V was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtA V did not result in cell elongation. CgtA V is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtA V protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.All living organisms express one or more Obg/CgtA GTPase. Elucidating the function of these GTPases has been complicated by somewhat contradictory phenotypes associated with specific mutants in different model systems. Some of the confusion stems from the dual function of these proteins (ribosome assembly and stress response) in at least some organisms, as well as from potential species-specific differences that may result in different phenotypes observed for orthologous obg/cgtA mutants. Perhaps the most surprising of these inconsistent reports is that of the nonessential nature of the Vibrio harveyi cgtA V gene (5) and its pleiotropic phenotypes (32,34,42), observations at odds with what has been reported for other obg/cgtA mutants. Here we show that, in contrast to these studies, the V. harveyi cgtA V gene is essential. Furthermore, we demonstrate that the trimethoprim resistance associated with a cgtA V clone was conferred by the linked folA gene. We also show that CgtA V is associated with the 50S ribosomal particle. From these studies, we predict that the role of CgtA V is similar to that of other bacterial Obg/CgtA proteins.Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were grown at 37°C (unless otherwise indicated) in Luria-Bertani media (LB; 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/liter) or LB agar (1.5% agar) containing antibiotics, as required (gentamicin, 30 g/ml; kanamycin, 30 g/ml; chloramphenicol, 10 g/ml; trimethoprim, 100 g/ml; ampicillin, 100 g/ml). V. harveyi strains were derived from BB7 (2) and maintained at 30°C on BOSS medium (10 g of Bacto Peptone, 3 g of beef extract, 30 g of NaCl, and 1 ml of 100% glycerol/liter) or BOSS agar (1.5% agar) supplemented with antibiotics (gentamicin, 30 g/ml; kanamycin, 50 g/ml).The V. harveyi cgtA V gene is essential. We were perplexed by the reported viability of the V. harveyi cgtA V transposon insertion mutant BB7X (5) for several reasons. First, in all other organisms examined, obg/cgtA is an essential gene (1,16,22,25,37,40). Second, given the sequence similarity among the Obg/CgtA proteins, we would predict that CgtA proteins play similar roles in all bacteria, and yet the viable nature and reported phenotypes of the V. harveyi cgtA V insertional mutant (5, 32, 34, 42) were different from those reported for other obg/cgtA mutant strains (7,16,17,22,26,38). Therefore, we reinvestigated the consequences of CgtA V depletion in V. harveyi. Plasmids used in the presen...

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Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

Cited by 40 publications

References 17 publications

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

TheVibrio harveyiGTPase CgtA_VIs Essential and Is Associated with the 50S Ribosomal Subunit

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Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

Cited by 40 publications

References 17 publications

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

Isolation of Dihydrofolate Reductase-Deficient Mutant of Escherichia coli

Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

TheVibrio harveyiGTPase CgtAVIs Essential and Is Associated with the 50S Ribosomal Subunit

Contact Info

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TheVibrio harveyiGTPase CgtA_VIs Essential and Is Associated with the 50S Ribosomal Subunit