Molecular analysis of 36 mutations at the mouse pink-eyed dilution (p) locus.

Abstract Thirty-six radiation- or chemically induced homozygous-lethal mutations at the p locus in mouse chromosome 7 have been analyzed at 17 loci defined by molecular probes to determine the types of lesions, numbers of p-region markers deleted or rearranged, regions of overlap of deletion mutations, and genetic distances between loci. A linear deletion map of the [Myod1, Ldh3]-[Snrpn, Znf127] region has been constructed from the molecular analyses of the p-locus deletions. The utility of these deletions as tools for the isolation and characterization of the genes specifying the neurological, reproductive, and developmental phenotypes genetically mapped to this region will grow as more detailed molecular analyses continue.

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Isolation and characterization of a serologically defined tumour membrane antigen from a chemically-induced rat sarcoma, HSN

International Journal of Cancer ◽

10.1002/ijc.2910470415 ◽

1991 ◽

Vol 47 (4) ◽

pp. 569-575 ◽

Cited By ~ 4

Author(s):

Olivier Léger ◽

Pamela L. Dunn ◽

B. Coles ◽

C. J. Dean

Keyword(s):

Isolation And Characterization ◽

Chemically Induced

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Isolation and characterization of conditional lethal mutations in the mot gene of bacteriophage T4

Journal of Molecular Biology ◽

10.1016/0022-2836(78)90058-x ◽

1978 ◽

Vol 126 (3) ◽

pp. 551-570 ◽

Cited By ~ 45

Author(s):

T. Mattson ◽

G. Van Houwe ◽

R.H. Epstein

Keyword(s):

Bacteriophage T4 ◽

Lethal Mutations ◽

Isolation And Characterization

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Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

10.1101/2020.06.22.166033 ◽

2020 ◽

Cited By ~ 4

Author(s):

Tongqing Zhou ◽

I-Ting Teng ◽

Adam S. Olia ◽

Gabriele Cerutti ◽

Jason Gorman ◽

...

Keyword(s):

Region Of Interest ◽

Molecular Probes ◽

Terminal Sequence ◽

Protease Cleavage ◽

Isolation And Characterization ◽

Cryo Electron Microscopy ◽

Specific Protease ◽

A Site ◽

Biotinylated Probes

SummaryBiotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

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Chemically-induced genetic damage in fish: Isolation and characterization of the dab (Limanda limanda) ras gene

Marine Pollution Bulletin ◽

10.1016/0025-326x(95)00132-7 ◽

1995 ◽

Vol 31 (4-12) ◽

pp. 457-459 ◽

Cited By ~ 5

Author(s):

J.M. Rotchell ◽

R.M. Stagg ◽

J.A. Craft

Keyword(s):

Genetic Damage ◽

Ras Gene ◽

Isolation And Characterization ◽

Chemically Induced

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Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants

Blood ◽

10.1182/blood-2010-03-267419 ◽

2010 ◽

Vol 116 (19) ◽

pp. 3944-3954 ◽

Cited By ~ 86

Author(s):

Keir M. Balla ◽

Geanncarlo Lugo-Villarino ◽

Jan M. Spitsbergen ◽

David L. Stachura ◽

Yan Hu ◽

...

Keyword(s):

Immune Response ◽

Peritoneal Cavity ◽

Chronic Exposure ◽

Transgenic Animal ◽

Adult Zebrafish ◽

Molecular Analyses ◽

Isolation And Characterization ◽

Parasitic Worms ◽

Prospective Isolation

Abstract Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2hi cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2hi cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

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Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

10.1101/2021.12.29.474491 ◽

2021 ◽

Author(s):

I-Ting Teng ◽

Alexandra F. Nazzari ◽

Misook Choe ◽

Tracy Liu ◽

Matheus Oliveira de Souza ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Serum Antibody ◽

Region Of Interest ◽

Molecular Probes ◽

Single Step ◽

Terminal Sequence ◽

Specific Amino Acid ◽

Protease Cleavage ◽

Isolation And Characterization

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

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