A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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Gene replacement with one-sided homologous recombination

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.360-367.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 360-367

Author(s):

N Berinstein ◽

N Pennell ◽

C A Ottaway ◽

M J Shulman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Target Gene ◽

Gene Replacement ◽

Immunoglobulin Genes ◽

Nonhomologous Recombination ◽

Immunoglobulin Locus ◽

Chromosomal Sequences ◽

Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

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The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells.

Genetics ◽

10.1093/genetics/136.2.597 ◽

1994 ◽

Vol 136 (2) ◽

pp. 597-605

Author(s):

A S Waldman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Insertion Mutant ◽

Insertion Mutation ◽

Homologous Pairing ◽

Rate Limiting Step ◽

Limiting Step ◽

Homology Searching ◽

Rate Limiting

Abstract Mouse LTK- cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.

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Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.74.10.4301 ◽

1977 ◽

Vol 74 (10) ◽

pp. 4301-4305 ◽

Cited By ~ 1

Author(s):

C. J. Collins ◽

J. T. Parsons

Keyword(s):

Dna Sequences ◽

Mammalian Cells ◽

Avian Sarcoma Virus ◽

Sarcoma Virus ◽

Avian Sarcoma

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Functional Studies of MLL-AF4 and a Murinized pSer-variant thereof: MLL-AF4 Impairs the Ribosome Biosynthesis Pathway

10.21203/rs.3.rs-743930/v1 ◽

2021 ◽

Author(s):

Anna Lena Siemund ◽

Eric Kowarz ◽

Rolf Marschalek

Keyword(s):

Transcription Factor ◽

Fusion Protein ◽

Mammalian Cells ◽

Biological Activities ◽

Intracellular Localization ◽

Biological Behavior ◽

Hematopoietic Stem ◽

Functional Studies ◽

Pol I ◽

Transcription Factor Complex

Abstract Background: Recent pathomolecular studies on the MLL-AF4 fusion protein revealed that the murinized version of MLL-AF4, the MLL-Af4 fusion protein, was able to induce leukemia when expressed in murine or human hematopoietic stem/progenitor cells (1). In parallel, a group from Japan demonstrated that the pSer domain of the AF4 protein, as well as the pSer domain of the MLL-AF4 fusion is able to bind the Pol I transcription factor complex SL1 (2).Here, we investigated the human MLL-AF4 and a pSer-murinized version thereof for their functional properties in mammalian cells. Gene expression profiling studies were complemented by intracellular localization studies and functional experiments concerning the biological activities in the nuecleolus.Results: Based on our results, we have to conclude that MLL-AF4 is predominantly localizing in the nucleolus, thereby interfering withPol I transcription, and subsequently,also ribosomebiogenesis. The murinized pSer-variant is more localizing in the nucleus, which may explain their different biological behavior. Of note, AF4-MLL is cooperating at the molecular level with MLL-AF4, but not with the pSer-murinized version of it.Conclusion: This study provides new insights and a molecular explanation for the known differences between hMLL-hAF4 (not leukemogenic) and hMLL-mAf4 (leukemogenic). While the human pSer domain is able to efficiently recruit the SL1 transcription factor complex, the murine counterpart is not. This has several consequences for our understanding of t(4;11) leukemia which is by far the most frequent leukemia in infants, childhood and adults suffering from MLL-r acute leukemia.

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Uptake, Fixation, and Expression of Foreign DNA in Mammalian Cells: The Organization of Integrated Adenovirus DNA Sequences

Current Topics in Microbiology and Immunology - Tumorviruses, Neoplastic Transformation and Differentiation ◽

10.1007/978-3-642-68654-2_6 ◽

1982 ◽

pp. 127-194 ◽

Cited By ~ 17

Author(s):

Walter Doerfler

Keyword(s):

Dna Sequences ◽

Mammalian Cells ◽

Foreign Dna

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The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus

Blood ◽

10.1182/blood.v83.5.1420.1420 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1420-1427 ◽

Cited By ~ 13

Author(s):

PK Sengupta ◽

DE Lavelle ◽

J DeSimone

Keyword(s):

Fusion Protein ◽

Developmental Stage ◽

Control Region ◽

Dna Sequences ◽

Nuclear Protein ◽

Globin Gene ◽

Locus Control Region ◽

Specific Factor ◽

Regulatory Sequences ◽

Gamma Globin

Abstract Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta- globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

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