[66] Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

Calzone, Frank J.; Britten, Roy J.; Davidson, Eric H.

doi:10.1016/0076-6879(87)52069-9

Cited by 213 publications

(85 citation statements)

References 13 publications

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“…Poly(A)' mRNA was isolated from mycelium grown for 20 h. Poly(A)+ mRNA (6 pg) and primer P12 (5'-TTCTTCAGCAAACCTCC-3') ( Fig. 2) (5 pg) were used for primer extension mapping, which was performed according to Calzone et al (1987) with minor modifications. The reaction products were analysed on a denaturing polyacrylamide gel parallel to a dideoxy sequence reaction of genomic pPKACl clone using primer P12.…”

Section: Reverse Transcription Pcr (Rt-pcr)mentioning

confidence: 99%

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

Benčina

Panneman²,

Ruijter³

et al. 1997

Microbiology

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The gene pkaC encoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungus Aspergillus niger. A probe for screening A. niger phage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) from A. niger has a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that - except for the first 15 amino acids, which are homologous to the Magnaporthe grisea PKA-C - shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A. niger shows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any other pkaC homologue in A. niger. The cloned pkaC was used for transformation of A. niger, leading to increased levels of pkaC mRNA and PKA-C activity. Transformants overexpressing pkaC were phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.

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Section: Reverse Transcription Pcr (Rt-pcr)mentioning

confidence: 99%

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

Benčina

Panneman²,

Ruijter³

et al. 1997

Microbiology

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“…The start site of the core I gene transcript was determined according to Calzone et al [19]. The oligonucleotide primer 5'-CTGGCACGCTCTGGAGAGCC-3' positioned 129 nucleotides from the 5' end of the pBCI l l clone was end labeled with [Y-~~PIATP (Amersham, 3 kCi/mmol) and T4 polynucleotide kinase (New England Biolabs), purified over a Sephadex G-50 column (Pharmacia) and annealed with 3 pg poly(A)-rich RNA from bovine heart for 3 h at 42°C.…”

Section: Primer Extensionmentioning

confidence: 99%

Core I protein of bovine ubiquinol–cytochrome‐c reductase; an additional member of the mitochondrial‐protein‐processing family

Gencic

Schägger

Jagow

1991

European Journal of Biochemistry

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Core I and core II proteins are the largest nuclear‐encoded subunits of the mitochondrial ubiquinol–cytochrome‐c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of λ ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase‐chain‐reaction‐amplified fragment. The core I precursor protein consists of 362 amino acids with a 34‐amino‐acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14‐amino‐acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter, E., Neupert, W. & Weiss, H. (1989) Nature 339, 147–149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol–cytochrome‐c reductase.

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“…5000 C/mmol, Amersham) and T4 polynucleotide kinase. Primer extension was made as described (Calzone et al, 1987) after annealing 15 fmol of oligonucleotide to poly(A) RNA from parental and hydroxyurea-resistant TA 3 cells at 50°C for 6 h. Total cellular RNA was extracted from logarithmically growing cells by the guanidium thiocyanate method (Chirgwin et al, 1979) and poly(A) RNA was recovered after one cycle of adsorption and elution from an oligodeoxythymidylate [oligo(dT)] -cellulose column (Aviv and Leder, 1972).…”

Section: Dna Sequencingmentioning

confidence: 99%

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Thelander

1989

The EMBO Journal

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[66] Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

Cited by 213 publications

References 13 publications

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

Core I protein of bovine ubiquinol–cytochrome‐c reductase; an additional member of the mitochondrial‐protein‐processing family

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

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