A single base change at a splice acceptor site leads to a truncated CAD protein in Urd?A mutant Chinese hamster ovary cells

1992 ◽  
Vol 18 (1) ◽  
pp. 65-75 ◽  
Author(s):  
David Patterson ◽  
Ralph Berger ◽  
John Bleskan ◽  
Diane Vannais ◽  
Jeffrey Davidson
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Base Change ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Ovary Cells ◽  
Single Base ◽  
Single Base Change ◽  
Cad Protein

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
Xiang-xing Zhu ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Biased Short Tract Repair of Palindromic Loop Mismatches in Mammalian Cells

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1257-1268
Author(s):  
Danielle G Taghian ◽  
Heather Hough ◽  
Jac A Nickoloff
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Single Base ◽  
Defective Mutant ◽  
Preferential Loss ◽  
In Cis ◽  
Mutant Cells

Abstract Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80–100%). When no other mismatches were present in hDNA, loops were retained with a 1.6–1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, ~25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
XIANGXING ZHU ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Denaturing Gradient Gel Analysis of Single-Base Substitutions at a MouseAdenine Phosphoribosyltransferase Splice Acceptor Site

1989 ◽  
Vol 2 (4) ◽  
pp. 217-225 ◽  
Author(s):  
Stephen R. Dlouhy ◽  
Dennis A. Schaff ◽  
James A. Trofatter ◽  
Hsiao-Sheng Liu ◽  
Peter J. Stambrook ◽  
...  
Keyword(s):  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Substitutions

scholarly journals Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand–receptor interaction

2007 ◽  
Vol 195 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Crystal Ying Wang ◽  
Yajun Wang ◽  
Amy Ho Yan Kwok ◽  
Frederick C Leung
Keyword(s):  
Ligand Binding ◽  
Splice Variants ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Splice Acceptor Site ◽  
Receptor Interaction ◽  
Acceptor Site

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp56) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH ≥10−8 M; PACAP ≥10−6 M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp56 is not a critical determinant for receptor activation and direct ligand–receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp56 by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp56 is nonessential for receptor activation and direct ligand–receptor interaction.

scholarly journals Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098 ◽  
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

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