Marker-fusion PCR for one-step mutagenesis of essential genes in yeast

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

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Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

Molecular Biotechnology ◽

10.1007/s12033-017-0050-7 ◽

2017 ◽

Vol 60 (2) ◽

pp. 92-99 ◽

Cited By ~ 2

Author(s):

Yilan Liu ◽

Jinjin Chen ◽

Anders Thygesen

Keyword(s):

Fusion Pcr ◽

One Step

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Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning

Biochimie ◽

10.1016/j.biochi.2020.04.002 ◽

2020 ◽

Vol 174 ◽

pp. 69-73

Author(s):

Libing Liao ◽

Lu Yang ◽

Yanxia Xu ◽

Xiaoli Li ◽

Guangxuan Tan ◽

...

Keyword(s):

Recombination Site ◽

Fusion Pcr ◽

One Step

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One Step Closer to Functionally Characterizing the Human Genome: Genomic Screens Identify Essential Genes and Liabilities in Cancer Cells

The Hematologist ◽

10.1182/hem.v13.2.4995 ◽

2016 ◽

Vol 13 (2) ◽

Author(s):

Justin Taylor ◽

Omar Abdel-Wahab

Keyword(s):

Cancer Cells ◽

Human Genome ◽

Essential Genes ◽

One Step

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Fusion PCR, a One-Step Variant of the “Megaprimer” Method of Mutagenesis

BioTechniques ◽

10.2144/98245bm08 ◽

1998 ◽

Vol 24 (5) ◽

pp. 736-742 ◽

Cited By ~ 23

Author(s):

Christiaan Karreman

Keyword(s):

Fusion Pcr ◽

One Step

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One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0051 ◽

2021 ◽

pp. mbc.E21-02-0051

Author(s):

Tsz Kwan Yeung ◽

Ho Wai Lau ◽

Hoi Tang Ma ◽

Randy Y.C. Poon

Keyword(s):

Gene Silencing ◽

Cell Lines ◽

Human Cell ◽

Mammalian Cells ◽

Essential Genes ◽

Gene Inactivation ◽

Human Cell Lines ◽

Efficient Generation ◽

Multiple Genes ◽

One Step

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.

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An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092670 ◽

1976 ◽

Vol 34 ◽

pp. 542-543

Author(s):

R.P. Goehner ◽

W.T. Hatfield ◽

Prakash Rao

Keyword(s):

Electron Diffraction ◽

Real Time ◽

Pattern Analysis ◽

Flow Diagram ◽

Hard Copy ◽

Time Analysis ◽

Real Time Analysis ◽

Transmission Electron ◽

One Step ◽

Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

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Nutrient-regulated gene expression in eukaryotes

Biochemical Society Symposium ◽

10.1042/bss0730085 ◽

2006 ◽

Vol 73 ◽

pp. 85-96 ◽

Cited By ~ 8

Author(s):

Richard J. Reece ◽

Laila Beynon ◽

Stacey Holden ◽

Amanda D. Hughes ◽

Karine Rébora ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Control ◽

Direct Interaction ◽

Signalling Pathways ◽

A Cell ◽

One Step ◽

Higher Eukaryotes ◽

Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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