Expression of chemically induced mutations in mammalian cells using an in situ procedure

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

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Construction and Characterization of Mutations within theKlebsiella mrkD1P Gene That Affect Binding to Collagen Type V

Infection and Immunity ◽

10.1128/iai.67.4.1672-1676.1999 ◽

1999 ◽

Vol 67 (4) ◽

pp. 1672-1676 ◽

Cited By ~ 7

Author(s):

Tricia A. Sebghati ◽

Steven Clegg

Keyword(s):

Collagen Type ◽

Gene Products ◽

Induced Mutations ◽

Binding Properties ◽

Collagen Binding ◽

Chemically Induced ◽

Collagen Type V ◽

Type V ◽

Type 3

ABSTRACT The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within themrkD 1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD 1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1Pprotein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.

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Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

10.1101/430264 ◽

2018 ◽

Author(s):

Marcin Leda ◽

Andrew J. Holland ◽

Andrew B. Goryachev

Keyword(s):

Symmetry Breaking ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Break Symmetry ◽

Biophysical Model ◽

Model Organisms ◽

Centriole Duplication ◽

Mother Centriole ◽

Autocatalytic Activation

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

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Detection of mRNA Transfer Between Mammalian Cells in Coculture by Single-Molecule Fluorescent In Situ Hybridization (smFISH)

Imaging Gene Expression - Methods in Molecular Biology ◽

10.1007/978-1-4939-9674-2_8 ◽

2019 ◽

pp. 109-129 ◽

Cited By ~ 1

Author(s):

Gal Haimovich ◽

Jeffrey E. Gerst

Keyword(s):

In Situ Hybridization ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent In Situ Hybridization

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Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures

Communications Biology ◽

10.1038/s42003-020-01174-y ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 2

Author(s):

Marc A. Beal ◽

Matthew J. Meier ◽

Danielle P. LeBlanc ◽

Clotilde Maurice ◽

Jason M. O’Brien ◽

...

Keyword(s):

Human Cancer ◽

Published Data ◽

Induced Mutations ◽

Lung Cancers ◽

Human Cancers ◽

Chemically Induced ◽

Mutation Pattern ◽

Environmental Causes ◽

Next Generation Sequencing Ngs

AbstractTransgenic rodent (TGR) models use bacterial reporter genes to quantify in vivo mutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation pattern analysis to inform mutational mechanisms. We used this approach to identify 2751 independent lacZ mutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706 lacZ mutations from eight additional environmental mutagens. We report that lacZ gene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a carcinogen present in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results suggest that the analysis of chemically induced mutations in the lacZ gene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and propose novel environmental causes of mutation signatures observed in human cancers.

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