Affinity Purification of Specific Chromatin Segments from Chromosomal Loci in Yeast

ABSTRACT Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states.

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A synthetic intron in a naturally intronless yeast pre-tRNA is spliced efficiently in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.329-331.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 329-331

Author(s):

M Winey ◽

I Edelman ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Analysis ◽

Single Copy ◽

Single Copy Gene ◽

Copy Gene

Saccharomyces cerevisiae glutamine tRNA(CAG) is encoded by an intronless, single-copy gene, SUP60. We have imposed a requirement for splicing in the biosynthesis of this tRNA by inserting a synthetic intron in the SUP60 gene. Genetic analysis demonstrated that the interrupted gene produces a functional, mature tRNA product in vivo.

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Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.

The Journal of Cell Biology ◽

10.1083/jcb.134.3.715 ◽

1996 ◽

Vol 134 (3) ◽

pp. 715-729 ◽

Cited By ~ 125

Author(s):

C Ruhrberg ◽

M A Hajibagheri ◽

M Simon ◽

T P Dooley ◽

F M Watt

Keyword(s):

Plasma Membrane ◽

Terminal Differentiation ◽

Human Keratinocytes ◽

Single Copy ◽

Epidermal Keratinocytes ◽

Single Copy Gene ◽

Cornified Envelope ◽

Keratin Filaments ◽

Copy Gene

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."

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CO2-Responsive CCT Protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes

10.22541/au.161704145.57069338/v1 ◽

2021 ◽

Author(s):

Hiroshi Fukayama ◽

Naoki Shibatani ◽

Hirofumi Miyagawa ◽

Aiko Koudou ◽

Yasuo Yamauchi ◽

...

Keyword(s):

Oryza Sativa L ◽

Large Subunit ◽

Starch Synthesis ◽

Gel Filtration ◽

Parenchyma Cell ◽

Mesophyll Cell ◽

Leaf Sheath ◽

Bimolecular Fluorescence Complementation ◽

Promoter Regions

CO2 responsive CCT protein (CRCT) is a positive regulator of starch synthesis related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5’-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa, however CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provide important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

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Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

10.1101/2019.12.12.874511 ◽

2019 ◽

Author(s):

Ruth A. Houseright ◽

Emily E. Rosowski ◽

Pui Ying Lam ◽

Sebastien JM Tauzin ◽

Oscar Mulvaney ◽

...

Keyword(s):

Gene Expression ◽

Bacterial Infections ◽

Large Scale ◽

Affinity Purification ◽

Neutrophil Migration ◽

Acute Injury ◽

Specific Gene ◽

Cell Type ◽

Cell Type Specific

AbstractFollowing acute injury, leukocytes rapidly infiltrate into tissues. For efficient recruitment, leukocytes must sense and respond to signals from both from the damaged tissue and from one another. However, the cell type specific transcriptional changes that influence leukocyte recruitment and wound healing have not been well characterized. In this study, we performed a large-scale translating ribosome affinity purification (TRAP) and RNA sequencing screen in larval zebrafish to identify genes differentially expressed by neutrophils, macrophages, and epithelial cells in the context of wounding. We identified the complement pathway and c3a.1, homologous to the C3A component of human complement, as significantly increased in neutrophils in response to a wound. We report that c3a.1−/− zebrafish larvae have impaired neutrophil responses to both tail wounds and localized bacterial infections, as well as increased susceptibility to infection due to a neutrophil-intrinsic function of C3A. We further show that C3A enhances migration of human primary neutrophils to IL-8 and that c3a.1−/− larvae have impaired neutrophil migration in vivo, and a decrease in neutrophil directed migration speed early after wounding. Together, our findings suggest a role for C3A in mediating efficient neutrophil migration to damaged tissues and support the power of TRAP to identify cell-specific changes in gene expression associated with wound-associated inflammation.

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Oligomerization of the influenza virus polymerase complex in vivo

Journal of General Virology ◽

10.1099/vir.0.83387-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 520-524 ◽

Cited By ~ 35

Author(s):

Núria Jorba ◽

Estela Area ◽

Juan Ortín

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Affinity Purification ◽

Significant Proportion ◽

Gel Filtration ◽

Agarose Gel ◽

Polymerase Complex ◽

Virus Transcription ◽

Transcription And Replication

The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.

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The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912409116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23618-23624 ◽

Cited By ~ 11

Author(s):

Audrey C. A. Cleuren ◽

Martijn A. van der Ent ◽

Hui Jiang ◽

Kristina L. Hunker ◽

Andrew Yee ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rna Sequencing ◽

Expression Profiles ◽

Affinity Purification ◽

Specific Gene ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing ◽

Vascular Beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

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A synthetic intron in a naturally intronless yeast pre-tRNA is spliced efficiently in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.329 ◽

1989 ◽

Vol 9 (1) ◽

pp. 329-331 ◽

Cited By ~ 2

Author(s):

M Winey ◽

I Edelman ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Analysis ◽

Single Copy ◽

Single Copy Gene ◽

Copy Gene

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Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0317 ◽

2013 ◽

Vol 368 (1616) ◽

pp. 20120317 ◽

Cited By ~ 31

Author(s):

Anke Wagner ◽

Lydia Segler ◽

Sabine Kleinsteuber ◽

Gary Sawers ◽

Hauke Smidt ◽

...

Keyword(s):

Transcriptional Response ◽

Single Copy ◽

Chlorinated Organic Compounds ◽

Promoter Regions ◽

Regulation Of Expression ◽

Reductive Dehalogenase ◽

Dehalococcoides Mccartyi ◽

Genes Encoding

The remarkable capacity of the genus Dehalococcoides to dechlorinate a multitude of different chlorinated organic compounds reflects the number and diversity of genes in the genomes of Dehalococcoides species encoding reductive dehalogenase homologues ( rdh ). Most of these genes are located in the vicinity of genes encoding multiple antibiotic resistance regulator (MarR)-type or two-component system regulators. Here, the transcriptional response of rdhA genes (coding for the catalytic subunit) to 2,3- and 1,3-dichlorodibenzo- p -dioxin (DCDD) was studied in Dehalococcoides mccartyi strain CBDB1. Almost all rdhA genes were transcribed in the presence of 2,3-DCDD, albeit at different levels as shown for the transcripts of cbrA , cbdbA1453, cbdbA1624 and cbdbA1588. By contrast, 1,3-DCDD did not induce rdhA transcription. The putative MarR CbdbA1625 was heterologously produced and its ability to bind in vitro to the overlapping promoter regions of the genes cbdbA1624 and cbdbA1625 was demonstrated. To analyse regulation in vivo , single-copy transcriptional promoter– lacZ fusions of different rdhA genes and of cbdbA1625 were constructed and introduced into the heterologous host Escherichia coli , and expression levels of the fusions were measured. The cbdbA1625 gene was cloned into a vector allowing a regulation of expression by arabinose and it was transformed into the strains containing the rdh -promoter– lacZ fusion derivatives. CbdbA1625 was shown to downregulate transcription from its own promoter resulting in a 40–50% reduction in the β-galactosidase activity, giving the first hint that it acts as a repressor.

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