A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

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Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1628-1634.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1628-1634

Author(s):

R Bookstein ◽

E Y Lee ◽

A Peccei ◽

W H Lee

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Long Range ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Line ◽

Cancer Cell Line ◽

Range Restriction ◽

Rb Gene ◽

Deletion Junction

Mutational inactivation of the retinoblastoma (RB) gene is considered a fundamental event in the formation of several types of human cancer. A substantial proportion of RB gene mutations are partial or complete deletions that extend an unknown distance beyond one or both ends of the gene. To provide a framework for measuring the extent of these deletions, we have constructed a long-range restriction map of SfiI sites spanning 850 kilobases around the RB gene. This map was applied in a molecular analysis of RB gene deletion in breast cancer cell line MB468. A previous study of this cell line demonstrated deletion of the entire RB gene except for exons 1 and 2 (E. Y.-H. P. Lee, H. To, J.-Y. Shew, R. Bookstein, P. Scully, and W.-H. Lee, Science 241:218-221, 1988). Genomic clones containing the deletion junction were isolated from a library made from MB468 DNA. A probe obtained from the far side of the deletion junction was used to localize and clone the unknown 3' endpoint, demonstrating that the chromosomal mutation in this case was a simple deletion spanning 200 kilobases. Sequence analysis of the deletion junction indicated a conservative deletion with no loss or gain of nucleotides. The deletion endpoints had no sequence homology to each other or to any repetitive sequence family, such as Alu, so the recombination event was illegitimate. Structural analysis of this and other RB gene deletions is important for understanding molecular mechanisms of recessive oncogenesis.

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Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5

Development ◽

10.1242/dev.126.4.839 ◽

1999 ◽

Vol 126 (4) ◽

pp. 839-849 ◽

Cited By ~ 18

Author(s):

J.M. Reecy ◽

X. Li ◽

M. Yamada ◽

F.J. DeMayo ◽

C.S. Newman ◽

...

Keyword(s):

Homeobox Gene ◽

Protein Isoforms ◽

Proximal Promoter ◽

Coding Region ◽

Heart Tube ◽

Regulatory Regions ◽

Regulatory Domains ◽

Exon 1 ◽

Flanking Sequences ◽

Upstream Exon

Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is activated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomach and tongue. One approach to elucidate the signals involved in cardiogenesis was to examine the transcriptional regulation of early lineage markers such as Nkx2-5. We generated F0 transgenic mice, which carry Nkx2-5 flanking sequences linked to a lacZ reporter gene. We identified multiple regulatory regions located within the proximal 10.7 kb of the Nkx2-5 gene. In addition to a proximal promoter, we identified a second promoter and a novel upstream exon that could participate in the regulation of Nkx2-5 transcription. Although used rarely in normal development, this novel exon could be spliced into the Nkx2-5 coding region in several ways, thereby potentially creating novel Nkx2-5 protein isoforms, whose transcriptional activity is greatly diminished as compared to wild-type Nkx2-5. An enhancer that directs expression in pharynx, spleen, thyroid and stomach was identified within 3.5 kb of exon 1 between the coding exon 1 and the novel upstream exon 1a. Two or more enhancers upstream of exon 1a were capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, then in the outflow tract and right ventricle of the looped heart tube. A negative element was also located upstream of exon1a, which interacted in complex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4. Our results demonstrate that a complex suite of interacting regulatory domains regulate Nkx2-5 transcription. Dissection of these elements should reveal essential features of cardiac induction and positive and negative signaling within the cardiac field.

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A Second Exon Splicing Silencer within Human Immunodeficiency Virus Type 1tatExon 2 Represses Splicing of Tat mRNA and Binds Protein hnRNP H

Journal of Biological Chemistry ◽

10.1074/jbc.m104070200 ◽

2001 ◽

Vol 276 (44) ◽

pp. 40464-40475 ◽

Cited By ~ 82

Author(s):

Sandrine Jacquenet ◽

Agnès Méreau ◽

Patricia S. Bilodeau ◽

Laurence Damier ◽

C. Martin Stoltzfus ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splicing Silencer ◽

Exon Splicing ◽

Immunodeficiency Virus

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HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer

Journal of Virology ◽

10.1128/jvi.01108-20 ◽

2020 ◽

Vol 94 (21) ◽

Author(s):

Ana Jordan-Paiz ◽

Maria Nevot ◽

Kevin Lamkiewicz ◽

Marie Lataretu ◽

Sandra Franco ◽

...

Keyword(s):

Protein Expression ◽

Codon Usage ◽

Virus Replication ◽

Synonymous Codon ◽

Replication Capacity ◽

Coding Region ◽

Cpg Dinucleotides ◽

Splicing Silencer ◽

Env Protein ◽

Hiv 1

ABSTRACT Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation. IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

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Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1628 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1628-1634 ◽

Cited By ~ 28

Author(s):

R Bookstein ◽

E Y Lee ◽

A Peccei ◽

W H Lee

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Long Range ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Line ◽

Cancer Cell Line ◽

Range Restriction ◽

Rb Gene ◽

Deletion Junction

Mutational inactivation of the retinoblastoma (RB) gene is considered a fundamental event in the formation of several types of human cancer. A substantial proportion of RB gene mutations are partial or complete deletions that extend an unknown distance beyond one or both ends of the gene. To provide a framework for measuring the extent of these deletions, we have constructed a long-range restriction map of SfiI sites spanning 850 kilobases around the RB gene. This map was applied in a molecular analysis of RB gene deletion in breast cancer cell line MB468. A previous study of this cell line demonstrated deletion of the entire RB gene except for exons 1 and 2 (E. Y.-H. P. Lee, H. To, J.-Y. Shew, R. Bookstein, P. Scully, and W.-H. Lee, Science 241:218-221, 1988). Genomic clones containing the deletion junction were isolated from a library made from MB468 DNA. A probe obtained from the far side of the deletion junction was used to localize and clone the unknown 3' endpoint, demonstrating that the chromosomal mutation in this case was a simple deletion spanning 200 kilobases. Sequence analysis of the deletion junction indicated a conservative deletion with no loss or gain of nucleotides. The deletion endpoints had no sequence homology to each other or to any repetitive sequence family, such as Alu, so the recombination event was illegitimate. Structural analysis of this and other RB gene deletions is important for understanding molecular mechanisms of recessive oncogenesis.

Download Full-text