Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments

Santangelo, George M.; Tornow, Joanne; Moldave, Kivie

doi:10.1016/0378-1119(86)90402-6

Cited by 10 publications

(9 citation statements)

References 13 publications

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“…We used a previously described cloning method to create a plasmid library of DNase I-generated small fragments of S. cerevisiae DNA (43). The In vivo assays were done after yeast colonies had been transferred from minimal agar to nitrocellulose filters.…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, some DNA sequences may activate transcription by virtue of their unique structural properties: poly(dA-dT) homopolymer tracts can function as bidirectional promoter elements in the yeast genome (48). These regions have a helix repeat of 10.0 base pairs (bp) instead of the normal 10.6 bp (38,40) and are associated with bends in DNA (28, 31).Of primary concern when attempting to analyze the yeast genome comprehensively by using promoter libraries is the large number of yeast colonies (approximately 106 [43]) that must be screened. The use of two reporter genes (divergently flanking the cloning site) should reduce that number by 50% relative to libraries that use a single reporter gene.…”

mentioning

confidence: 99%

“…Of primary concern when attempting to analyze the yeast genome comprehensively by using promoter libraries is the large number of yeast colonies (approximately 106 [43]) that must be screened. The use of two reporter genes (divergently flanking the cloning site) should reduce that number by 50% relative to libraries that use a single reporter gene.…”

mentioning

confidence: 99%

“…We created a comprehensive S. cerevisiae genomic library in pPCO (43) by inserting random fragments into the adhllac4 intergenic region. Filter colony assays were used to detect either ADH or P-galactosidase activity in yeast trans-formants.…”

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confidence: 99%

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Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Santangelo¹,

Tornow²,

McLaughlin³

et al. 1988

Mol. Cell. Biol.

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Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or I-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region.Transcription of several well-studied genes in Saccharomyces cerevisiae is thought to require at least two cisacting regions of DNA: the proximal TATA element (12,23,32,34) and the distal upstream activating sequence (UAS;9,14,23,25,45,46,50). UASs are bidirectional elements: they function even when inverted (22,36,47). Transcription does not always require the presence of a TATA element; efficient expression of the PGK gene depends on UASPGK but does not require TATA sequences (36). Unlike mRNA synthesis in higher eucaryotes, sequences in the vicinity of each mRNA start site contribute to the specificity of transcription initiation (12,32,34). Interestingly, some DNA sequences may activate transcription by virtue of their unique structural properties: poly(dA-dT) homopolymer tracts can function as bidirectional promoter elements in the yeast genome (48). These regions have a helix repeat of 10.0 base pairs (bp) instead of the normal 10.6 bp (38,40) and are associated with bends in DNA (28, 31).Of primary concern when attempting to analyze the yeast genome comprehensively by using promoter libraries is the large number of yeast colonies (approximately 106 [43]) that must be screened. The use of two reporter genes (divergently flanking the cloning site) should reduce that number by 50% relative to libraries that use a single reporter gene. A vector that contains two reporter genes permits the detection of a unidirectional promoter regardless of its orientation in the cloning site. Also, if a promoter is bidirectional, it can be isolated from the library in a configuration that allows the simultaneous monitoring of transcription in both directions along the DNA helix. With this in mind, we have used a promoter-cloning vector (pPCO; Fig. 1 uninterrupted coding region. The 53-bp upstream intergenic region lacks a UAS, poly(dA-dT) tract, or TATA element.We created a comprehensive S. cerevisiae genomic library in pPCO (43) by inserting random fragments into the adhllac4 intergenic region. Filter colony assays were used to detect eithe...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Santangelo¹,

Tornow²,

McLaughlin³

et al. 1988

Mol. Cell. Biol.

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“…1-kbp EeoRI-BglII or EeoRI-BamHI fragments were substituted for corresponding fragments on pSMF63 and pSMF38T, to construct pSMF102 and pSMF103, respectively. Random cloning of short DNA fragments prepared from yeast chromosomal DNA was carried out essentially as described by Santangelo et al (1986).…”

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confidence: 99%

Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Nishizawa

Ozawa

Hishinuma

1989

Appl Microbiol Biotechnol

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Two kinds of yeast secretion vectors were constructed by site-directed mutagenesis of the invertase signal sequence and ligation of synthetic oligonucleotides coding appropriate signals. Each has a cloning site for a foreign gene preceded by a sequence encoding either the signal peptide cleavage site or a Lys-Arg sequence which is a cleavage site for the product of the KEX2 gene. Both vectors were able to direct the expression and secretion of mouse amylase. One of them has a Sall site within the signal sequence, and an attempt to clone sequences enhancing secretion of amylase with this vector is reported.tase, which could be unfavourable for proper functioning of the product. To prevent the synthesis of the extension, vectors with a cloning site either at the signal cleavage site or at a position following a Lys-Arg sequence, which is a target site of the KEX2 gene product (KEX2p; Julius et al. 1984a), were constructed by site-directed mutagenesis and ligation of appropriate synthetic oligonucleotides. These vectors were able to direct the production and secretion of a foreign protein. The vectors proved useful for the study of factors affecting secretion efficiency.

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Facilitating functional analysis of theSaccharomyces cerevisiae genome using anEGFP-based promoter library and flow cytometry

Bell¹,

Davies

Attfield

1999

Yeast

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A promoter library was generated to facilitate identification of differentially regulated promoters in Saccharomyces cerevisiae. The library was constructed in a vector containing two reporter genes (EGFP and lacZ) divergently arranged about a unique cloning site. Approximately 2×105 clones were obtained and a flow cytometer was used to screen the library for copper‐induced EGFP expression. A DNA fragment conferring copper‐inducible expression of EGFP was rapidly identified. This DNA fragment, which contained several motifs associated with copper and oxidative stress homeostasis, lies upstream of two ‘orphan’ genes of unknown function. Further studies comparing expression from episomal vs. integrative vectors showed that construction of a similar library using an integrative vector would further enhance rapid identification of genes that are differentially regulated in S. cerevisiae. The ability to identify regulated promoters rapidly should facilitate the functional analysis of the yeast genome by identifying genes induced by specific physiological conditions. Copyright © 1999 John Wiley & Sons, Ltd.

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Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments

Cited by 10 publications

References 13 publications

Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Facilitating functional analysis of theSaccharomyces cerevisiae genome using anEGFP-based promoter library and flow cytometry

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