scholarly journals Changes in the size of cell nuclei in response to SMC proteins degradation in mammalian cells

2021 ◽  
Keyword(s):  
Mammalian Cells ◽  
Cell Nuclei ◽  
Smc Proteins

scholarly journals Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 59-69 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Recombinant Dna ◽  
Dna Duplexes ◽  
Dna Molecules ◽  
Cell Nuclei ◽  
High Concentration ◽  
Length Polymorphisms

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.

Pre-mRNA splicing and nuclear organization

1992 ◽  
Vol 50 (1) ◽  
pp. 502-503
Author(s):  
David L. Spector ◽  
Gayle Lark ◽  
Sui Huang
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Transformed Cells ◽  
Splicing Factors ◽  
Cell Nuclei ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
Passage Number ◽  
Immortal Cells ◽  
Labeling Pattern

Several major classes of small nuclear ribonucleoprotein particles (snRNPs) (Ul, U2, U4/U6, U5) as well as other splicing factors have been shown to be involved in the processing of pre-mRNA molecules. For most RNA polymerase II transcripts, such processing includes the addition of a 7-methylguanosine cap structure at the 5’ end of the nascent RNA transcripts, hnRNP assembly, splicing, polyadenylation, and the exchange of hnRNP proteins for mRNP proteins. Splicing of nuclear pre-mRNA molecules occurs in spliceosomes, macromolecular complexes composed of a pre-mRNA, snRNPs and other splicing factors. Since RNA processing is essential to cellular function, we and others have been interested in identifying the organization of splicing factors in cell nuclei. We have examined the localization of snRNPs in a variety of mammalian cells and have observed differences in the organization of these factors in transformed cells, immortal cells, and cells of defined passage number. Cells of defined passage number exhibit a speckled staining pattern after immunolabeling with anti-Sm, anti-B’ or anti-m3G antibodies. Furthermore, 2 to 3% of the cells, in a given population, exhibit 1 to 2 round “foci” in addition to the speckled labeling pattern. However, transformed cells exhibited 1 to 4 intensely stained round foci, in 81 to 99% of the cells, in addition to the speckled labeling pattern. Immortal cells exhibited 1 to 4 intensely stained smaller foci in 4 to 40% of the cells, in addition to the speckled labeling pattern. When immortal cells (REF-52) which had been transformed by adenovirus (REF-52 Ad5.4) were examined, these cells exhibited an increase in the percentage of cells containing 1 to 2 intensely stained foci, in addition to the speckled labeling, from 24% to 99%. We have identified these intensely stained foci as coiled bodies which can be visualized in the nucleoplasm of cells with or without antibody labeling. This study is the first to directly correlate an increase in the number of cells containing coiled bodies in a given cell population with the transformed phenotype. Based on this study, we conclude that the organization of snRNPs within the mammalian cell nucleus is a reflection of the physiology of the cell which may change upon transformation or immortalization.

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific

1994 ◽  
Vol 14 (1) ◽  
pp. 170-180
Author(s):  
A L Nicolás ◽  
C S Young
Keyword(s):  
Mammalian Cell ◽  
Mammalian Cells ◽  
Nuclear Extract ◽  
Major Effect ◽  
Cell Nuclei ◽  
Site Specific ◽  
Preferential Formation ◽  
Nuclear Extracts ◽  
Dna Substrates ◽  
Molecular And Biochemical Mechanisms

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei

1985 ◽  
Vol 5 (1) ◽  
pp. 70-74
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Kanamycin Resistance ◽  
Linear Plasmid ◽  
Wild Type ◽  
Cell Nuclei ◽  
Heteroduplex Dna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
A Cell

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

scholarly journals Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

1994 ◽  
Vol 14 (1) ◽  
pp. 170-180 ◽  
Author(s):  
A L Nicolás ◽  
C S Young
Keyword(s):  
Mammalian Cell ◽  
Mammalian Cells ◽  
Nuclear Extract ◽  
Major Effect ◽  
Cell Nuclei ◽  
Site Specific ◽  
Preferential Formation ◽  
Nuclear Extracts ◽  
Dna Substrates ◽  
Molecular And Biochemical Mechanisms

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

scholarly journals Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 70-74 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Kanamycin Resistance ◽  
Linear Plasmid ◽  
Wild Type ◽  
Cell Nuclei ◽  
Heteroduplex Dna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
A Cell

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

Depletion in nuclear spermine during human spermatogenesis, a natural process of cell differentiation

1992 ◽  
Vol 263 (2) ◽  
pp. C343-C347 ◽  
Author(s):  
V. Quemener ◽  
Y. Blanchard ◽  
D. Lescoat ◽  
R. Havouis ◽  
J. P. Moulinoux
Keyword(s):  
Cell Proliferation ◽  
Cell Differentiation ◽  
Mammalian Cells ◽  
Gradient Centrifugation ◽  
Cell Nuclei ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Human Spermatogenesis

Polyamines (PA), polycations present in all mammalian cells, are essential for cell proliferation and differentiation. In vitro, PA are known to bind to DNA with a high affinity. In vivo, the intimate association of endogenous PA with highly condensed chromatin has been reported. During spermatogenesis, when processes of cell proliferation and differentiation take place, the potential role of polyamines has not been studied in depth. We report here the PA levels measured in human spermatogenic cell nuclei at different stages of differentiation. Cell populations (spermatocytes and round, elongating, or elongated spermatids) were obtained after submitting human testes to a trypsin-deoxyribonuclease digestion, then to a centrifugal elutriation and Percoll gradient centrifugation. A significant and progressive nuclear spermine level decrease was observed from primary spermatocytes to elongated spermatids. This release of spermine from nuclei was concomitant with three major events in mammalian spermiogenesis: the reduction of DNA transcription activity, the replacement of histone proteins by protamines, and the compaction of chromatin. This is the first report arguing a release of nuclear spermine during an in vivo physiological cell differentiation process.

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

scholarly journals Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10088-10101 ◽  
Author(s):  
Rodion Gorchakov ◽  
Natalia Garmashova ◽  
Elena Frolova ◽  
Ilya Frolov
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Protein Complexes ◽  
Nonstructural Proteins ◽  
Cell Nuclei ◽  
High Concentration ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Rna Complexes

ABSTRACT Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

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