Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.
The Escherichia coli RecR protein participates in a recombinational DNA repair process. Its gene is located in a region of chromosome that extends from 502 to 509 kilobases on the physical map and that contains apt, dnaX, orfl2-recR, htpG, and adk. Most, if not all, of these are involved in nucleic acid metabolism. The orfl2-recR reading frames consist of 935 base pairs and overlap by one nucleotide, with the 3' A of the orfl2 termination codon forming the 5' nucleotide of the recR initiation codon. The orfl2-recR promoter was located upstream of orfl2 by sequence analysis, promoter cloning, and S1 nuclease protection analysis. The start point of transcription was determined by primer extension. The transcript 5' end contained a long, apparently untranslated region of 199 nucleotides. Absence of a detectable promoter specific for recR and the overlap of the orfl2 and recR reading frames suggest that translation of recR is coupled to that of orfl2. By maxicell analysis, it was determined that both orfp2 and recR are translated.Escherichia coli recR mutants were identified by Mahdi and Lloyd (20) as derivatives of a recB sbcB sbcC strain which became recombination deficient and UV sensitive. recR mutations reduced recombination after conjugation or transduction in a recBC sbcBC background but had little effect in a recBC+ sbcBC+ background (20). recR mutations also increased UV sensitivity, but in both recBC sbcBC and recBC+ sbcBC+ strains. It was concluded, therefore, that the RecR product participates in a recombinational repair pathway (20). The fact that recR mutations decreased recombination proficiency and UV repair when combined as recB recR but not as recF recR double mutants indicated that recR is part of the RecF pathway (20). Although the recR mutation had little effect on recombination after conjugation or transduction in an otherwise wild-type strain, it did cause deficiency in plasmid recombination (20).The recR gene was mapped near min 11 (20), clockwise of and near the DNA replication gene dnaX (16,22). We report here that the region between dnaX and the nearby htpG gene (3) contains two overlapping reading frames which encode proteins of 12 and 22 kilodaltons (kDa). The first reading frame is designated orfl2 in accordance with the proposal of Mahdi and Lloyd (21), who also sequenced this region. (Their report appeared while this manuscript was in preparation.) The second is the recR gene, as shown also by Mahdi and Lloyd (21).The orfl2 and recR frames overlap by one nucleotide pair, suggesting translational coupling, and the promoter which expresses both is located upstream of orfl2 within the dnaX coding sequence. MATERIAL AND METHODSBacterial strains, plasmids, and bacteriophages. The E. coli K-12 strains are listed in Table 1. pBJ1 is a 6.2-kilobase-pair * Corresponding author.
One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G 1 -S and G 2 -M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G 1 -S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G 2 -M and G 1 -S CDKs in human cells is essentially quantitative.
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