A simple and very efficient method for generating cDNA libraries

Gubler, Ueli; Hoffman, Beth J.

doi:10.1016/0378-1119(83)90230-5

Cited by 4,503 publications

(1,605 citation statements)

References 13 publications

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“…'/@) mice and from their normal littermates (cch/cch or cc%?). cDNA libraries in lgtl0 (Huynh et al, 1985) were constructed following the protocol by Gubler and Hoffman (1983) et al, 1988). The newborn cf"/cch mouse liver cDNA library (average insert size 1.6 kb) contained 2 x lo7 recombinants of which 5 x lo5 pfu were amplified on Escherichia coli C600hfl.…”

Section: Constructionmentioning

confidence: 99%

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

Ruppert

Boshart

Bosch

et al. 1990

Cell

142

105

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Section: Constructionmentioning

confidence: 99%

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

Ruppert

Boshart

Bosch

et al. 1990

Cell

142

105

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“…Specific and random primed cDNA libraries were constructed in hgtl0 according to [13]. 2 pg poly(A)+ RNA yielded 5 x lo6 independent recombinant phages.…”

Section: Construction and Screening Of Cdna Librariesmentioning

confidence: 99%

The cDNA of the two isoforms of bovine cGMP‐dependent protein kinase

Wernet¹,

Flockerzi²,

Hofmann³

1989

FEBS Letters

187

128

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cDNAs encoding the isoform Ia of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in IgtlO. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 420742181. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform IB of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.

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“…For this purpose, a Agtl0 cDNA library was constructed from poly(A) ÷ RNA of the trunk of rainbow trout embryos. The double strands cDNAs synthetized by the method of Gubler et al [11] were size fractionated by gel filtration on a sepharose 4B column (Pharmacia) and the largest fractions were pooled, inserted into Agtl0 vector (Stratagene) and encapsided using an in vitro packaging kit (Amersham). After amplification of the cDNA library, approximately 5 • 105 plaques were screened at low stringency with a fragment from the Xenopus MyoD cDNA encompassing the B-HLH domain [12].…”

mentioning

confidence: 99%

Identification of a muscle factor related to MyoD in a fish species

Rescan

Gauvry

Pabœuf

et al. 1994

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.Key words: MyoD; Myogenesis; Teleost; (Satellite cell)The MyoD gene identified by substract cloning for myoblast specific RNA is the prototype of a family of master regulators of skeletal myogenesis which includes in vertebrates three other members namely myogenin, Myf5 and MRF4 identified subsequently [1][2][3][4][5]. All these genes encode proteins that share a highly conserved central region termed the basic/helix-loop-helix(B-HLH) domain related to the c-myc superfamily and contain sequences essential for both dimerisation and DNA binding [6]. In multipotential 10T1/2 cells, transfection experiments have shown that forced expression of these exogenous myogenic factors is sufficient to drive them down the muscle differentiation pathway suggesting their functions in myogenic lineage determination [1][2][3][4][5]. In contrast to vertebrates whose genome encodes multiple members of the MyoD family, invertebrates, including Sea urchin [7], C. elegans [8] and Drosophila [9], appear to contain only a single myogenic factor encoding gene. However, the myogenic factor for Sea urchin and C. elegans activates myogenesis in 10T1/2 cells indicating a highly conserved mechanism for muscle genes activation. Myogenic factors have been studied in mammals, amphibians and birds [10], but nothing is known to date in fish. To analyse early developmental events leading to muscle formation in fish, we set out to isolate myogenic regulatory factors from Rainbow trout (Oncorhynchus mykiss) embryos (398 degree days) using a probe which spanned functional domains of the Xenopus MyoD cDNA. For this purpose, a Agtl0 cDNA library was constructed from poly(A) ÷ RNA of the trunk of rainbow trout embryos. The double strands cDNAs synthetized by the method of Gubler et al. [11] were size fractionated by gel filtration on a sepharose 4B column (Pharmacia) and the largest fractions were pooled, inserted into Agtl0 vector (Stratagene) and encapsided using an in vitro packaging kit (Amersham). After amplification of the cDNA library, approximately 5 • 105 plaques were screened at low stringency with a fragment from the Xenopus MyoD cDNA encompassing the B-HLH domain [12]. From 9 positive clones, we identified a single cDNA of 1.5 kb which had two EcoRI fragments of aproximately 1.2 and 0.3 kb. Restriction analysis and sequencing showed that this internal EcoRI site was not situated near a Notl site which is contained in the linker...

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A simple and very efficient method for generating cDNA libraries

Cited by 4,503 publications

References 13 publications

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes

The cDNA of the two isoforms of bovine cGMP‐dependent protein kinase

Identification of a muscle factor related to MyoD in a fish species

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