Construction of a uniform-abundance (normalized) cDNA library.

Patanjali, Sankhavaram R.; Parimoo, Satish; Weissman, Sherman M.

doi:10.1073/pnas.88.5.1943

Cited by 151 publications

(100 citation statements)

References 31 publications

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“…If this third possibility explains the results of the screen, it suggests that many more bHLH proteins which are expressed at other stages or in a smaller fraction of the embryo may exist. These bHLH proteins would be missed in this screen but could be identified by using a library from a different stage of development or a specific cell type or by screening a normalized library (44). In support of this, two-hybrid screens of heart and teratoma libraries with da have identified additional new bHLH proteins (31,39).…”

Section: Discussionmentioning

confidence: 89%

Identification of a New Family of Tissue-Specific Basic Helix-Loop-Helix Proteins with a Two-Hybrid System

Hollenberg

Sternglanz

Cheng

et al. 1995

Molecular and Cellular Biology

633

564

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With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage-and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.During development, specific genetic programs are activated by transcription factors which are often stage and tissue specific in their distribution. Understanding the combinatorial code of factors which defines each cell fate during the differentiation process requires identification of new factors with restricted spatial activity. One group of factors with pivotal roles during development are the basic helix-loop-helix (bHLH) proteins. These factors are important in differentiation processes as diverse as skeletal myogenesis, neurogenesis, and hematopoiesis (for reviews, see references 2, 12, 13, 43, 59, and 63).bHLH proteins have been grouped into four classes based on partner choice, DNA-binding ability, and transcriptional activity. (i) E proteins, also called class A, are widely expressed and act as partners for at least two of the other classes (6,42,47). (ii) Tissue-specific bHLH proteins, or class B, pair with E proteins to produce heterodimers. Class A homodimers and A/B heterodimers are thought to function as transcriptional activators (9,46,62). (iii) Id proteins lack a basic region and form a non-DNA-binding complex with class A and some class B proteins (6, 55). (iv) Hairy and Enhancer-of-split [h/E(spl)] bHLH proteins form a fourth class whose interactions with the other three groups are not well characterized. The h/E(spl) family is structurally defined, in part, by a WRPW motif at the C terminus and an absolutely conserved proline in the basic region. These proteins act genetically as repressors of bHLHmediated functions and can bind to DNA v...

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Section: Discussionmentioning

confidence: 89%

Identification of a New Family of Tissue-Specific Basic Helix-Loop-Helix Proteins with a Two-Hybrid System

Hollenberg

Sternglanz

Cheng

et al. 1995

Molecular and Cellular Biology

633

564

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“…Approximately 90% of 700 clones sequenced from the luciferasederived shRNA library contained proper shRNA structures comprising unique 19-21-bp sequences of the luciferase gene. To generate a human transcriptome library and to overcome the problem of uneven representation of different genes in conventional cDNA, we took advantage of the process of cDNA normalization that equalizes the abundance of different mRNA sequences (21). As the starting material for shRNA library construction, we used a library originally developed for the selection of genetic suppressor elements (GSE, short cDNA fragments encoding either antisense RNA or protein fragments acting as transdominant inhibitors).…”

mentioning

confidence: 99%

“…As the starting material for shRNA library construction, we used a library originally developed for the selection of genetic suppressor elements (GSE, short cDNA fragments encoding either antisense RNA or protein fragments acting as transdominant inhibitors). The GSE library was derived from randomly fragmented cDNA of MCF7 human breast carcinoma cells, normalized by C o t fractionation (21). The GSE library construction strategy (22) favored directional cloning of cDNA fragments flanked by different adaptors at the 5′ and 3′ ends relative to the original mRNA; sequence analysis of representative clones showed that approximately two thirds of the clones contained the adaptors in the preferred orientation.…”

mentioning

confidence: 99%

Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing

Shtutman¹,

Maliyekkel

Shao

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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As a general strategy for function-based gene identification, an shRNA library containing ≈150 shRNAs per gene was enzymatically generated from normalized (reduced-redundance) human cDNA. The library was constructed in an inducible lentiviral vector, enabling propagation of growth-inhibiting shRNAs and controlled activity measurements. RNAi activities were measured for 101 shRNA clones representing 100 human genes and for 201 shRNAs derived from a firefly luciferase gene. Structure-activity analysis of these two datasets yielded a set of structural criteria for shRNA efficacy, increasing the frequencies of active shRNAs up to 5-fold relative to random sampling. The same library was used to select shRNAs that inhibit breast carcinoma cell growth by targeting potential oncogenes. Genes targeted by the selected shRNAs were enriched for 10 pathways, 9 of which have been previously associated with various cancers, cell cycle progression, or apoptosis. One hundred nineteen genes, enriched through this selection and represented by two to six shRNAs each, were identified as potential cancer drug targets. Short interfering RNAs against 19 of 22 tested genes in this group inhibited cell growth, validating the efficiency of this strategy for high-throughput target gene identification.

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“…The primers from this kit add adapters and Sfi cloning sites to the cDNA fragments. Normalization was done as described (Patanjali et al, 1991). Briefly, PCR-amplified cDNA was heat-denatured and allowed to reanneal for 24, 48, 72 or 96 h. Single-stranded and double-stranded DNA from each time point was separated by hydroxyapatite chromatography.…”

Section: Preparation Of Normalized Gse Cdna Librarymentioning

confidence: 99%

Falkor, a novel cell growth regulator isolated by a functional genetic screen

et al. 2002

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A novel cell growth regulator, named Falkor, was identified using a functional approach to mammalian gene cloning, the Genetic Supressor Elements (GSE) method. In this screen, expression of the C-terminal domain of Falkor conferred cells with resistance to cisplatin-induced growth arrest. Expression of the Cterminus of Falkor, but not of the full-length protein, enhanced cell growth both following genotoxic stress and under normal conditions suggesting a general role for this protein in cell growth control. This effect of the Cterminus fragment was abrogated by over-expression of the full-length Falkor, suggesting that the fragment counteracts the function of the full-length protein. Falkor is encoded by a 2-kb mRNA which is present at different levels in various tissues, and is localized in the nucleus of cells. The C-terminal domain of Falkor, isolated from the GSE library, has significant homology to a known human and rat cell growth regulator, SM-20, and to the C. elegans protein EGL-9, recently shown to modify the Hypoxia Inducible Factor-1a. The homology suggests that these proteins share a functional domain that is conserved among a family of growth regulation proteins.

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Construction of a uniform-abundance (normalized) cDNA library.

Cited by 151 publications

References 31 publications

Identification of a New Family of Tissue-Specific Basic Helix-Loop-Helix Proteins with a Two-Hybrid System

Identification of a New Family of Tissue-Specific Basic Helix-Loop-Helix Proteins with a Two-Hybrid System

Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing

Falkor, a novel cell growth regulator isolated by a functional genetic screen

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