Suppressor of Hairless is required for signal reception during lateral inhibition in the Drosophila pupal notum

Development ◽  
1995 ◽  
Vol 121 (6) ◽  
pp. 1875-1884 ◽  
Author(s):  
F. Schweisguth
Keyword(s):  
Gene Dosage ◽  
Sense Organ ◽  
Cell Analysis ◽  
Relative Level ◽  
Suppressor Of Hairless ◽  
H Gene ◽  
Proneural Cluster ◽  
Mutant Cells ◽  
H Protein ◽  
Selection Of

Suppressor of Hairless (Su(H)) activity is zygotically required in larval imaginal discs for the singling out of adult sense organ precursor (SOP) cells: loss of Su(H) function results in too many proneural cluster cells adopting the SOP fate, while overexpression of the Su(H) protein prevents SOP specification. Su(H) null mutant alleles are recessive lethal at the late larval and early pupal stages. The development of Su(H) mutant cells in pupae was therefore studied in somatic clones. Clonal analysis first showed that Su(H) is required for the regular spacing of microchaete precursor cells, as clusters of mutant SOPs were detected at positions where singled out sense organ cells are normally found. Second, Su(H) mutant SOPs produced neuron-like cells, consistent with a late defect in Notch (N) signalling. Third, a careful cell-by-cell analysis of clone borders showed that Su(H) mutant cells may adopt the SOP fate even when directly adjacent to wild-type cells. Finally, quantitative clone border analysis indicates that the relative level of Su(H) gene dosage appears to bias the selection of the future SOP: cells with a higher level of Su(H) activity are more likely to adopt the epidermal fate. These results show that notum cells strictly require Su(H) activity for receiving the lateral inhibitory signal. Thus, the DNA-binding protein encoded by the Su(H) gene may act downstream of the N receptor to implement the epidermal, non-SOP fate.

scholarly journals Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Hiroshi Sato ◽  
Hiroki Kato ◽  
Haruyoshi Yamaza ◽  
Keiji Masuda ◽  
Huong Thi Nguyen Nguyen ◽  
...  
Keyword(s):  
Normal Cell ◽  
Expression Vector ◽  
Gene Dosage ◽  
Genetic Disorders ◽  
Human Cells ◽  
Chromosome 21 ◽  
Disease Phenotypes ◽  
Simplex Virus ◽  
Selection Of ◽  
Trisomic Cell

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

scholarly journals Bovine Plasma β-Mannosidase Activity and Its Potential use for β-Mannosidosis Carrier Detection

1992 ◽  
Vol 4 (4) ◽  
pp. 434-440 ◽  
Author(s):  
Kevin T. Cavanagh ◽  
Margaret Z. Jones ◽  
Bruce Abbitt ◽  
Ronald Skinner
Keyword(s):  
Enzyme Activity ◽  
Gene Dosage ◽  
Low Risk ◽  
Carrier Detection ◽  
Biological Factors ◽  
Gene Dosage Effect ◽  
Bovine Plasma ◽  
The Mean ◽  
Potential Use ◽  
Selection Of

Plasma β-mannosidase activities were determined for Salers cattle from 8 herds as an evaluation of this method for detection of β-mannosidosis heterozygotes. Several biological factors, such as age, gender, herd, and risk of being a β-mannosidosis carrier, were considered in this study. The mean enzyme activity for obligate heterozygotes (n = 8) was 55 U/ml (range = 43–65 U/ml), which was 59% of the mean enzyme activity for cattle that were low risk for being a carrier. These data indicate that bovine β-mannosidosis is characterized by a gene dosage effect. The analytical and biological variation of plasma β-mannosidase activity that was observed necessitates limiting the test to adult fullblood/purebred Salers cattle within a herd. Plasma β-mannosidase analysis provides important information for intraherd selection of Salers cattle that are heterozygous for β-mannosidosis.

scholarly journals Probing neutralizing-antibody responses against emerging measles viruses (MVs): immune selection of MV by H protein-specific antibodies?

2005 ◽  
Vol 86 (2) ◽  
pp. 365-374 ◽  
Author(s):  
Sabine Santibanez ◽  
Stefan Niewiesk ◽  
Alla Heider ◽  
Jürgen Schneider-Schaulies ◽  
Guy A. M. Berbers ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Measles Virus ◽  
Protein A ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Neutralizing Capacity ◽  
Human Sera ◽  
H Protein ◽  
Neutralizing Epitopes ◽  
Selection Of

Measles virus (MV) infection and vaccination induce long-lasting immunity and neutralizing-antibody responses that are directed against the MV haemagglutinin (H) and the fusion (F) protein. A new MV genotype, D7, emerged recently in western Germany and rapidly replaced the long-term endemically circulating genotypes C2 and D6. Analysis of the H gene of C2, D6, D7 and vaccine viruses revealed uniform sequences for each genotype. Interestingly, a consistent exchange of seven distinct amino acids in the D7 H was observed when compared with residues shared between C2, D6 and vaccine viruses, and one exchange (D416→N) in the D7 H was associated with an additional N-linked glycosylation. In contrast, the F gene is highly conserved between MVs of these genotypes. To test whether the D7 H protein escapes from antibody responses that were raised against earlier circulating or vaccine viruses, the neutralizing capacity of mAbs recognizing seven distinct domains on the H of an Edmonston-related MV was compared. The mAbs revealed a selective and complete loss of two neutralizing epitopes on the D7 H when compared with C2, D6 and vaccine viruses. To assess whether these alterations of the D7 H affect the neutralizing capacity of polyclonal B-cell responses, genotype-specific antisera were produced in cotton rats. However, no significant genotype-dependent difference was found. Likewise, human sera obtained from vaccinees (n=7) and convalescents (n=6) did not distinguish between the MV genotypes. Although the hypothesis of selection of D7 viruses by pre-existing neutralizing antibodies is compatible with the differing pattern of neutralizing epitopes on the H protein, it was not confirmed by the results of MV neutralization with polyclonal sera.

scholarly journals A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa ◽  
Kuoyuan Cheng ◽  
Mark D. M. Leiserson ◽  
Prashant Jain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Selective Advantage ◽  
Driver Mutations ◽  
Cancer Driver ◽  
Oncogenic Mutation ◽  
Genome Wide ◽  
Selection For ◽  
Mutant Cells ◽  
Selection Of

AbstractRecent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.

scholarly journals On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy

Micromachines ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 561
Author(s):  
Meltem Elitas
Keyword(s):  
Antibiotic Resistance ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Single Cell Analysis ◽  
Binding Protein ◽  
Cell Analysis ◽  
Penicillin Binding Protein ◽  
Intact Cells ◽  
The Impact ◽  
Mutant Cells

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1673-1682 ◽  
Author(s):  
M. Gho ◽  
M. Lecourtois ◽  
G. Geraud ◽  
J.W. Posakony ◽  
F. Schweisguth
Keyword(s):  
Subcellular Localization ◽  
Cell Fate ◽  
Sense Organ ◽  
Wing Disc ◽  
Tissue Polarity ◽  
Cell Fate Decisions ◽  
Suppressor Of Hairless ◽  
Evolutionarily Conserved ◽  
Disc Cells ◽  
Sensory Organ Precursor

During imaginal development of Drosophila, Suppressor of Hairless [Su(H)], an evolutionarily conserved transcription factor that mediates intracellular signalling by the Notch (N) receptor, controls successive alternative cell fate decisions leading to the differentiation of multicellular sensory organs. We describe here the distribution of the Su(H) protein in the wing disc epithelium throughout development of adult sense organs. Su(H) was found to be evenly distributed in the nuclei of all imaginal disc cells during sensory organ precursor cells selection. Thus differential expression and/or subcellular localization of Su(H) is not essential for its function. Soon after division of the pIIa secondary precursor cell, Su(H) specifically accumulates in the nucleus of the future socket cell. At the onset of differentiation of the socket cell, Su(H) is also detected in the cytoplasm. In this differentiating cell, N and deltex participate in the cytoplasmic retention of Su(H). Still, Su(H) does not colocalize with N at the apical-lateral membranes. These observations suggest that N regulates in an indirect manner the cytoplasmic localization of Su(H) in the socket cell. Finally, the pIIb, shaft and socket cells are found to adopt invariant positions along the anteroposterior axis of the notum. This raises the possibility that tissue-polarity biases these N-mediated cell fate choices.

asense is a Drosophila neural precursor gene and is capable of initiating sense organ formation

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 1-17 ◽  
Author(s):  
M. Brand ◽  
A.P. Jarman ◽  
L.Y. Jan ◽  
Y.N. Jan
Keyword(s):  
Ectopic Expression ◽  
Sense Organ ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Gene Products ◽  
Proneural Gene ◽  
Proneural Genes ◽  
Helix Loop Helix ◽  
Normal Gene ◽  
Proneural Cluster

Neural precursor cells in Drosophila arise from the ectoderm in the embryo and from imaginal disc epithelia in the larva. In both cases, this process requires daughterless and the proneural genes achaete, scute and lethal-of-scute of the achaete-scute complex. These genes encode basic helix-loop-helix proteins, which are nuclear transcription factors, as does the asense gene of the achaete-scute complex. Our studies suggest that asense is a neural precursor gene, rather than a proneural gene. Unlike the proneural achaete-scute gene products, the asense RNA and protein are found in the neural precursor during its formation, but not in the proneural cluster of cells that gives rise to the neural precursor cell. Also, asense expression persists longer during neural precursor development than the proneural gene products; it is still expressed after the first division of the neural precursor. Moreover, asense is likely to be downstream of the proneural genes, because (1) asense expression is affected in proneural and neurogenic mutant backgrounds, (2) ectopic expression of asense protein with an intact DNA-binding domain bypasses the requirement for achaete and scute in the formation of imaginal sense organs. We further note that asense ectopic expression is capable of initiating the sense organ fate in cells that do not normally require the action of asense. Our studies therefore serve as a cautionary note for the inference of normal gene function based on the gain-of-function phenotype after ectopic expression.

scholarly journals Current techniques for single-cell lysis

2008 ◽  
Vol 5 (suppl_2) ◽  
Author(s):  
Robert B Brown ◽  
Julie Audet
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Laser Pulses ◽  
High Temporal Resolution ◽  
Cell Analysis ◽  
Careful Selection ◽  
Analysis Techniques ◽  
Cell Lysate ◽  
Selection Of

Owing to the small quantities of analytes and small volumes involved in single-cell analysis techniques, manipulation strategies must be chosen carefully. The lysis of single cells for downstream chemical analysis in capillaries and lab-on-a-chip devices can be achieved by optical, acoustic, mechanical, electrical or chemical means, each having their respective strengths and weaknesses. Selection of the most appropriate lysis method will depend on the particulars of the downstream cell lysate processing. Ultrafast lysis techniques such as the use of highly focused laser pulses or pulses of high voltage are suitable for applications requiring high temporal resolution. Other factors, such as whether the cells are adherent or in suspension and whether the proteins to be collected are desired to be native or denatured, will determine the suitability of detergent-based lysis methods. Therefore, careful selection of the proper lysis technique is essential for gathering accurate data from single cells.

scholarly journals Chromosomal Integration of tcbChlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics

2000 ◽  
Vol 66 (8) ◽  
pp. 3255-3261 ◽  
Author(s):  
Michael Klemba ◽  
Barbara Jakobs ◽  
Rolf-Michael Wittich ◽  
Dietmar Pieper
Keyword(s):  
Carbon Source ◽  
Gene Cluster ◽  
Gene Dosage ◽  
Single Copy ◽  
Degradation Pathway ◽  
Kanamycin Resistance ◽  
Chromosomal Integration ◽  
The Relationship ◽  
Subsequent Selection ◽  
Selection Of

ABSTRACT The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway inPseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying thetcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of thetcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.

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