Abstract：
:We have developed a simple, effective genetic screen for mutant alleles of eukaryotic DNA topoisomerase I that manifest severely depressed or complete loss of enzymatic function. The screen is based on the extreme toxicity of vaccinia topoisomerase expression in the Escherichia coli lysogen strain BL21(DE3) and is notable for its ease in distinguishing nonsense mutations (that result in truncated proteins) from missense mutations. The power of the method is evinced by our observation that 100% of the candidate alleles identified in the screen were ultimately found to have single-base changes at the DNA level that result in amino acid substitutions at the protein level. By mutagenizing plasmid DNA in vitro with hydroxylamine and applying this phenotypic screen, we have isolated five distinct single amino acid substitution mutants, each of which shows a biochemical phenotype, that is, greater than or equal to 90% reduction in specific DNA relaxing activity of the mutant protein relative to wild type. The amino acids thus implicated in topoisomerase function have identical or related counterparts at homologous positions in the topoisomerases from yeast and man. The same genetic screen has been applied to the selection of temperature-sensitive alleles of the vaccinia topoisomerase, leading to the isolation of two additional single-hit mutant alleles that display a temperature-sensitive growth phenotype in E. coli BL21(DE3). By broadening our mutagenesis procedures, we expect to generate a comprehensive map of vaccinia topoisomerase function and primary protein structure that should have direct application to eukaryotic cellular enzymes. Our methodology should be applicable to the selection of missense and conditional mutant alleles in other genes whose expression in bacteria is toxic.
journal_name
Genes Devjournal_title
Genes & developmentauthors
Morham SG,Shuman Sdoi
10.1101/gad.4.4.515subject
Has Abstractpub_date
1990-04-01 00:00:00pages
515-24issue
4eissn
0890-9369issn
1549-5477journal_volume
4pub_type
杂志文章abstract：:U6 small nuclear RNA is one of the spliceosomal RNAs involved in pre-mRNA splicing. In the fission yeast Schizosaccharomyces pombe, the U6 RNA gene was found to have an intron similar to a nuclear pre-mRNA intron, and it was proposed that the U6 intron might be inserted erroneously during pre-mRNA splicing. Using the ...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.5.6.1022
更新日期：1991-06-01 00:00:00
abstract：:Pre-mRNA splicing is essential for generating mature mRNA and is also important for subsequent mRNA export and quality control. The splicing history is imprinted on spliced mRNA through the deposition of a splicing-dependent multiprotein complex, the exon junction complex (EJC), at approximately 20 nucleotides upstrea...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.260403
更新日期：2003-04-15 00:00:00
abstract：:Developmental responses to the plant hormone auxin are thought to be mediated by interacting pairs from two protein families: short-lived inhibitory IAA proteins and ARF transcription factors binding to auxin-response elements. monopteros mutants lacking activating ARF5 and the auxin-insensitive mutant bodenlos fail t...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.229402
更新日期：2002-07-01 00:00:00
abstract：:The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1664408
更新日期：2008-07-15 00:00:00
abstract：:Spindle checkpoint silencing is a critical step during mitosis that initiates chromosome segregation, yet surprisingly little is known about its mechanism. Protein phosphatase I (PP1) was shown recently to be a key player in this process, and in this issue of Genes & Deverlopment, Akiyoshi and colleagues (pp. 2887-289...
journal_title：Genes & development
pub_type： 评论,杂志文章,评审
doi：10.1101/gad.1882109
更新日期：2009-12-15 00:00:00
abstract：:YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivatio...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.262816.115
更新日期：2015-06-15 00:00:00
abstract：:The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin ...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.12.13.2073
更新日期：1998-07-01 00:00:00
abstract：:RNAi has revolutionized reverse genetics; however, RNAi is not necessarily ubiquitous or constitutive. Lund and colleagues (pp. 1121-1131) show that microRNA (miRNA) effector Argonautes (Agos) are limiting and easily saturated during early Xenopus embryogenesis. Moreover, this stage is devoid of slicing capacity. Supp...
journal_title：Genes & development
pub_type： 评论,杂志文章
doi：10.1101/gad.2062911
更新日期：2011-06-01 00:00:00
abstract：:Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displa...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.189274.112
更新日期：2012-06-01 00:00:00
abstract：:We have identified protein kinase C-zeta (PKC-zeta) as a novel suppressor of neoplastic transformation caused by the v-raf oncogene. PKC-zeta overexpression drastically retards proliferation, abolishes anchorage-independent growth, and reverts the morphological transformation of v-raf-transformed NIH-3T3 cells. The mo...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.10.12.1455
更新日期：1996-06-15 00:00:00
abstract：:The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pr...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1657308
更新日期：2008-07-01 00:00:00
abstract：:Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studie...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.193789.112
更新日期：2012-08-15 00:00:00
abstract：:Chromosomal translocations are a hallmark of hematopoietic malignancies. The initial molecular events or pathways that lead to translocations in hematopoietic cells are largely unknown, particularly in the stem cell-enriched population postulated to be the initial target for these events. We used in vitro differentiat...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1522807
更新日期：2007-05-01 00:00:00
abstract：:The mechanisms by which homeoproteins bind selectively to target genes in vivo have long remained unresolved. Here we report that PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein by regulating its subnuclear localization and proximity to target genes. We demonstrate that the interaction of Msx1 with PIAS...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1392006
更新日期：2006-04-01 00:00:00
abstract：:The homeless (hls) gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. At a low frequency, females homozygous for mutations in hls generate early egg chambers in which the oocyte is positioned incorrectly within the cyst. At a high frequency, late-stage egg chambers exh...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.9.20.2495
更新日期：1995-10-15 00:00:00
abstract：:Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and m...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.188292.112
更新日期：2012-09-01 00:00:00
abstract：:Expression of HNF-4, a transcription factor in the steroid hormone receptor superfamily, is detected only in the visceral endoderm of mouse embryos during gastrulation and is expressed in certain embryonic tissues from 8.5 days of gestation. To examine the role of HNF-4 during embryonic development, we disrupted the g...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.8.20.2466
更新日期：1994-10-15 00:00:00
abstract：:Activated transcription by RNA polymerase II (Pol II) requires coactivators, one of which is the SRB/mediator. Whereas Srb4, an essential subunit of the SRB/mediator, is broadly required for Pol II transcription in yeast, we have shown that it is dispensable for the transcriptional activation of some genes. Here, we s...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.13.22.2934
更新日期：1999-11-15 00:00:00
abstract：:Intron homing in phage T4 occurs in the context of recombination-dependent replication, by virtue of intron-encoded endonucleolytic activity. After the td intron endonuclease I-TevI cleaves the intronless recipient 23 and 25 nucleotides upstream of the intron insertion site, exonucleolytic degradation is required for ...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.10.17.2158
更新日期：1996-09-01 00:00:00
abstract：:TFIID is a multisubunit protein containing the TATA box-binding polypeptide (TBP) and associated factors (TFIID-TAFs) required for activated transcription by RNA polymerase II. TBPs from different eukaryotes contain a highly conserved carboxy-terminal domain and very divergent amino-terminal domains. Earlier studies p...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.7.2.180
更新日期：1993-02-01 00:00:00
abstract：:Evidence is presented that the activation of the RNA polymerase sigma factor sigma(W) in Bacillus subtilis by regulated intramembrane proteolysis is governed by a novel, membrane-embedded protease. The sigma(W) factor is activated by proteolytic destruction of the membrane-bound anti-sigma(W) factor RsiW in response t...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1440606
更新日期：2006-07-15 00:00:00
abstract：:Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.2.8.1047
更新日期：1988-08-01 00:00:00
abstract：:Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy demands after birth. PPARgamma coactivator-1 (PGC-1) alpha and beta have been implicated in the transcriptional control of cellular energy metabolism. Mice with combined deficiency of PGC-1alpha and PGC-1beta ...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1661708
更新日期：2008-07-15 00:00:00
abstract：:For all newborn mammals, mother's milk is the perfect nourishment, crucial for their postnatal development. Here we report that, unexpectedly, maternal western diet consumption in mice causes the production of toxic milk that contains excessive long chain and saturated fatty acids, which triggers ceramide accumulation...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.191031.112
更新日期：2012-06-15 00:00:00
abstract：:Genomic imprinting, an inherently epigenetic phenomenon defined by parent of origin-dependent gene expression, is observed in mammals and flowering plants. Genome-scale surveys of imprinted expression and the underlying differential epigenetic marks have led to the discovery of hundreds of imprinted plant genes and co...
journal_title：Genes & development
pub_type： 杂志文章,评审
doi：10.1101/gad.269902.115
更新日期：2015-12-15 00:00:00
abstract：:The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promote...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.1698108
更新日期：2008-10-15 00:00:00
abstract：:The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) coactivator complex exerts functions in gene expression, including activator interaction, histone acetylation, histone deubiquitination, mRNA export, chromatin recognition, and regulation of the basal transcription machinery. These diverse functions involve distinct modu...
journal_title：Genes & development
pub_type： 杂志文章,评审
doi：10.1101/gad.184705.111
更新日期：2012-03-15 00:00:00
abstract：:We report the discovery of mRNA 5'-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5'-leader apparently derived from a 46-nucleotide RNA that shares features with previously charact...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.865401
更新日期：2001-02-01 00:00:00
abstract：:How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these seq...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.177428.111
更新日期：2012-01-01 00:00:00
abstract：:DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in...
journal_title：Genes & development
pub_type： 杂志文章
doi：10.1101/gad.299198.117
更新日期：2017-05-15 00:00:00
