Identification of a cullin homology region in a subunit of the anaphase- promoting complex

Science

Volumn 279, Issue 5354, 1998, Pages 1219-1222

  Yu, Hongtao ^a   Peters, Jan Michael ^a   King, Randall W ^a   Page, Andrew M ^a   Hieter, Philip ^a   Kirschner, Marc W ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CULLIN; CYCLIN DEPENDENT KINASE INHIBITOR; CYCLINE; PROTEIN SUBUNIT; UBIQUITIN PROTEIN LIGASE; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ANAPHASE; ARTICLE; HUMAN; NONHUMAN; PRIORITY JOURNAL; PROTEIN ANALYSIS; SACCHAROMYCES CEREVISIAE; SEQUENCE HOMOLOGY;

AMINO ACID SEQUENCE; ANAPHASE; ANIMALS; CELL CYCLE; CELL CYCLE PROTEINS; CLONING, MOLECULAR; CULLIN PROTEINS; HELMINTH PROTEINS; HUMANS; LIGASES; MOLECULAR SEQUENCE DATA; MUTATION; OPEN READING FRAMES; PHYLOGENY; PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SEQUENCE ALIGNMENT; UBIQUITIN-PROTEIN LIGASE COMPLEXES; UBIQUITIN-PROTEIN LIGASES;

SACCHAROMYCES CEREVISIAE;

EID: 0032549116     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5354.1219     Document Type: Article

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23. To clone APC5, we searched the EST database at the National Center for Biotechnology Information with the sequence of peptide PK123 (Fig. 1A) with BLAST and found that EST 309267 encodes amino acid sequences 90% identical to PK123. Primers that match the DNA sequence of the EST were used to isolate a full-length clone (APC5-H19) from a human testis plasmid library with the Gene Trapper system (GIBCO BRL) by the manufacturer's protocols. For the cloning of APC7, EST 40875 and EST 41898 were found to encode sequences 81% identical to peptide PK133. Primers that match the sequences of these ESTs were used to isolate APC7-H1 from the human testis library. Because no full-length clones were obtained, the 5′ 300-base pair (bp) region of clone APC7-H1 was amplified by PCR and used as a probe to isolate the full-length clone APC7-H18 from a HeLa Uni-ZAP cDNA library (Stratagene). On the basis of the Xenopus peptide sequences, multiple ESTs were identified for CDC23. EST 452573 contained the longest 5′ sequence and was ordered from the American Type Culture Collection. The 5′ 300-bp region of this EST was then amplified by PCR and used as a probe to isolate the full-length clone CDC23-H1 from the HeLa cDNA library. For the cloning of APC2, primers were designed from the sequence of EST 136462 that was identified by the peptides. These primers were used to clone a 1.3-kb fragment (APC2-H1) of the human APC2 cDNA from the testis library with the Gene Trapper system. The 5′ end of APC2-H1 was used as a probe to screen the HeLa library, resulting in the isolation of a 2.1-kb clone, APC2-H7. The 5′ end of APC2-H7 was then amplified and used to isolate the full-length clone APC2-H19 from a human breast cancer cDNA library (MDA468). To clone APC4, we identified a human EST (98516) that encodes sequences 87% identical to one of the APC4 peptides (PK108). Primers corresponding to the 5′ and 3′ regions of this EST were used to amplify a 300-bp DNA fragment from HeLa cDNA (Clontech, Palo Alto, CA) by PCR. This DNA fragment was then used to isolate the full-length clone APC4-H2 from the MDA468 library.
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30. A 4.5-kb Cla I to Eco RI fragment containing YLR127c was subcloned from cosmid 9233 into pRS316 to generate pAP1, which was sufficient to restore the viability of apc2::HIS3 spores, A cassette containing the URA3 gene flanked by triple HA repeats and sequences identical to the desired site of integration was amplified from pMPY-3XHA by PCR and transformed into a wild-type haploid strain carrying pAP3 (TRP1/CEN/APC2). Plasmid DNA was recovered from URA3 revertants, retransformed into Escherichia coli, and digested. Plasmids containing the URA3 insertion were retransformed into YPH499, and Ura3 revertants were selected on medium containing 5-fluoroorotic acid (5FOA). Extracts from cultured revertants were screened by immunoblotting with the 12CA5 antibody to HA for the presence of a specific band with the expected size.
31. The HIS3 gene was amplified with a primer pair containing a sequence flanking the YLR127c ORF. The PCR product was transformed into a diploid wildtype strain (YPH501) and selected for on synthetic complete (SC)-HiS plates. For His-positive transformants, replacement of YLR127C was confirmed by Southern (DNA) blotting with two independent probes.
32. A 3-kb cassette containing the entire APC2 ORF flanked by about 300 bp of noncoding sequence including the promoter was amplified by PCR and cloned into pRS314. Clones that rescued YAP11, an APC2 plasmid shuffle strain (an apc2::HIS3 mutant, rescued by pAP1, a CEN/URA3/APC2 plasmid), were used as templates for PCR mutagenesis. We used primers that hybridized to pRS vector sequence flanking the multicloning site to amplify a 3.7-kb cassette and cotransformed it with Bam HI-linearized pRS315 into YAP11 or YAP13. After the wild-type copy of APC2 was shuffled out by growth on 5FOA, transformants were replica-plated twice to SC-Leu and grown at either 25° or 37°. Plasmid DNA was recovered from transformants that successfully retested for temperature sensitivity. A cassette containing the mutant apc2 allele under its own promoter was subcloned into a LEU2 integrating vector and transformed into YAP11 or YAP13. Leu-positive transformants were assayed for their ability to lose the wild-type APC2 URA3 plasmid on 5FOA. The 5FOA-resistant colonies were then screened for their ability to recapitulate the temperature sensitivity and for complementation by episomal wild-type APC2.
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34. We thank L. Lum and C. Blobel for providing the human MDA468 cDNA library; D. Koshland for the antibody to Pds1p; G. Fang, B.-B. Zhou, and C. Gieffers for helpful discussions; and W. Zachariae and K. Nasmyth for communicating results before publication. H.Y. is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (grant DRG-1340). J.-M.P. was the recipient of a European Molecular Biology Organization fellowship. A.P. is a predoctoral student in the Biochemistry Cellular and Molecular Biology training program at the Johns Hopkins University School of Medicine. This research is supported by NIH grants CA16519 to P.H. and GM39023-08 and GM26875-17 to M.W.K.