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Volumn 288, Issue 5469, 2000, Pages 1248-1251

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE PHOSPHATE; PROTEIN KINASE;

EID: 0034685949     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5469.1248     Document Type: Article
Times cited : (643)

References (41)
  • 8
    • 0342770686 scopus 로고    scopus 로고
    • +). The glycogen content in muscle was determined as in (4-6). The CriMap software (28) was used for linkage analysis, and the Chrompic option was used to identify unlikely double recombinants. Genotyping of the R200Q mutation in PRKAG3 indicated that the recombinants (involving RN only) were caused by errors in the deduced RN genotype; the RN phenotype test is not 100% accurate
    • +). The glycogen content in muscle was determined as in (4-6). The CriMap software (28) was used for linkage analysis, and the Chrompic option was used to identify unlikely double recombinants. Genotyping of the R200Q mutation in PRKAG3 indicated that the recombinants (involving RN only) were caused by errors in the deduced RN genotype; the RN phenotype test is not 100% accurate. Details on previously described genetic markers are available online at PigBase www.ri.bbsrc.ac.uk/ pigmap/pigbase/pigbase.html. The genetic markers developed in this project will be described elsewhere, and a table with marker information is available from the authors.
  • 9
    • 0342770687 scopus 로고    scopus 로고
    • note
    • Radiation hybrid (RH) mapping was performed with the IMpRH panel (29) and the RHMAP 3.0 software (30).
  • 10
    • 0343205337 scopus 로고    scopus 로고
    • Sequence contigs constructed with PHRAP (31-33) were masked for repeats using RepeatMasker
    • Sequence contigs constructed with PHRAP (31-33) were masked for repeats using RepeatMasker, available online at http://ftp.genome.washington.edu/cgi-bin/ RepeatMasker.
  • 12
    • 0343640947 scopus 로고    scopus 로고
    • The NCBI nucleotide database is available online at www.ncbi.nlm.nih.gov.
  • 15
    • 0342335746 scopus 로고    scopus 로고
    • Online Mendelian Inheritance in Man database, March 2000. Available at www.ncbi.nlm.nih.gov/Omim/.
  • 16
    • 0343640946 scopus 로고    scopus 로고
    • note
    • 2-terminal region and a different sequence in the COOH-terminal end. Our data suggest that alternative splicing occurs in the 5′ region of the gene and experimental data are needed to determine which of the ATG codon or codons are used for initiation of translation. In regards to the difference in the COOH-terminal end, we believe that the one reported here is the correct one because it is consistent with the human genomic sequence as well as the pig coding sequence.
  • 18
    • 0343640926 scopus 로고    scopus 로고
    • The domain structure was assessed with the Pfam database, available online at www.cgr.ki.se/Pfam/. CBS domains are found in a wide range of proteins (see www.sanger.ac.uk/Users/agb/CBS/CBS.html).
  • 19
    • 0343640944 scopus 로고    scopus 로고
    • note
    • Human multiple tissue Northern blots (Clontech, Palo Alto, CA) were hybridized with human PRKAG1 (IMAGE clone 0362755; GenBank AA018675), human PRKAG2 (IMAGE clone 0322735; GenBank W15439), and porcine PRKAG3 (including codons 189 to 410) probes with ExpressHybridization solution (Clontech). Filters were washed with 0.1× standard saline citrate (SSC) and 0.1% SDS at 50°C and were exposed to film overnight.
  • 21
    • 0343205335 scopus 로고    scopus 로고
    • note
    • A 259 bp fragment of pig PRKAG3 including codon 200 was PCR-amplified with forward (5′-GGAGCAAATGTGCAGACAAG-3′) and reverse (5′-CCCACGAAGCTCTGCTTCTT-3′) primers. The oligonucleotide ligation assay (OLA) (34) was used for allele discrimination at nucleotide position 599. Each 10-μl OLA reaction contained 0.5 pmol of the two allele-specific probes (5′Hex-TGGCCAACGGCGTCCA-3′, 5′ROX-GGCCAACGGCGTCCG-3′) and the common probe (5′phosphate-AGCGGCACCTTTGTGAAAAAAAAAA-3′), 1.5 U of thermostable Ampligase and reaction buffer (Epicentre Technologies, Madison, WI), and 0.5 μl of the PCR product. After an initial incubation at 95°C for 5 min, the thermocycling profile was repeated 10 times as follows: denaturation at 94°C for 30 s and probe annealing and ligation at 55°C for 90 s. After OLA cycling, 1 μl of product was heat-denatured at 94°C for 3 min, cooled on ice, and loaded onto a sequencing gel.
  • 37
    • 0343205331 scopus 로고    scopus 로고
    • Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
    • Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  • 38
    • 0342335743 scopus 로고    scopus 로고
    • note
    • -) pigs. Muscle extract was purified up to and including the DEAE-Sepharose ion-exchange step as described (35). AMPK activity was assayed by phosphorylation of a synthetic peptide, HMRSAMSGLHLVKRR (36, 37). Assays were performed in triplicate for each animal in the presence and in the absence of 200 μM AMP. Because 0.1 μg of total protein from the 0.2 M NaCl DEAE eluates showed a linear activity both in the presence and absence of added AMP for at least 12 min (D. Milan et al., data not shown), we used this amount of extract and an incubation time of 10 min in the experiments. For each assay, we included controls that lacked added peptide. The values presented represent peptide-dependent activity.
  • 41
    • 0342335742 scopus 로고    scopus 로고
    • note
    • We thank E. Bongcam-Rudloff, J. C. Caritez, I. Hansson, K. Lilja, S. Marklund, and A. Törnsten for valuable assistance; J. Naveau, P. Cherel, and C. Lecour for pig DNA; R. Erlandsson and B. Amini for sequence analysis of BAC 127G6 at the Genome Center, Royal Institute of Stockholm, Technology and the UK-Human Genome Mapping Program Resource Centre for providing IMAGE clones. Supported by INRA, French Commisariat à l'Energie Atomique, Swedish Research Council for Forestry and Agriculture, Swedish Natural Science Research Council, Swedish Meat Marketing Board, Swedish Foundation for Strategic Research, the EC-funded project FarmChip (BIO4-CT98-0285), and Deutsche Forschungsgemeinschaft (Lo 689/1-1).


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