Conserved initiator proteins in eukaryotes

Science

Volumn 270, Issue 5242, 1995, Pages 1667-1671

  Gavin, Kimberley A ^a,b   Hidaka, Masumi ^a   Stillman, Bruce ^a  

^a Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

^b STONY BROOK UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; DNA BINDING PROTEIN; FUNGAL PROTEIN; PROTEIN SUBUNIT;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARTICLE; CAENORHABDITIS ELEGANS; CELL CYCLE PARAMETERS; DNA REPLICATION; DNA REPLICATION ORIGIN; EUKARYOTE; HUMAN; HUMAN CELL; KLUYVEROMYCES; MOLECULAR CLONING; NONHUMAN; PITYROSPORUM ORBICULARE; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; SCHIZOSACCHAROMYCES POMBE;

EID: 0029586090     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.270.5242.1667     Document Type: Article

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31. PCR reactions were carried out with K. lactis genomic DNA and degenerate oligonucleotides ORC1-3N (5′-AC[A/C/T]TATTC[A/C/T]GC[A/C/T]TATATGAT[A/C/ T] CA-3′) and ORC1-2C(5′-AAGTTC[T/G/A]GC[T/G/ A] GT[T/C]AA[G/A]TA[T/C]AA[T/G]TC-3′) based on regions of conservation between Orc1p and Sir3p. A 230-base pair (bp) amplified product related to S. cerevisiae Orc1p was used as a probe to screen a K. lactis genomic plasmid library [M. J. R. Stark and J. S. Milner, Yeast 5, 35 (1989)]. Two positive clones were isolated and were sequenced on both strands by standard dideoxy sequencing. The GenBank accession number is SU40151.
32. + was used as a probe. The same probe was used to screen an ordered genomic cosmid library [T.Mizukami et al., Cell 73, 121 (1993)]. Cosmid DNA was isolated from positive clones, and hybridizing fragments were subcloned and sequenced on both strands with the use of dideoxy sequencing. A 2.0-kb Eco RI fragment was used as a probe to screen a cDNA library [J. Field et al., Mol. Cell Biol. 8, 2159 (1988)]. Positive clones were plaque-purified, and DNA was isolated and sequenced on both strands. The GenBank accession number is U40378.
33. + was used as a probe. The same probe was used to screen an ordered genomic cosmid library [T.Mizukami et al., Cell 73, 121 (1993)]. Cosmid DNA was isolated from positive clones, and hybridizing fragments were subcloned and sequenced on both strands with the use of dideoxy sequencing. A 2.0-kb Eco RI fragment was used as a probe to screen a cDNA library [J. Field et al., Mol. Cell Biol. 8, 2159 (1988)]. Positive clones were plaque-purified, and DNA was isolated and sequenced on both strands. The GenBank accession number is U40378.
34. Reverse transcription of human 293 total RNA was performed with degenerate primer PO1PCR7 (5′-[C/T]TCIGGIAG[A/G]TCCATIGT[A/G]TT-3′). The resulting cDNA was used as a template in PCR reactions containing primers PO1PCR7 and PO1PCR3 (5′-GT[C/G/T]CG[C/G/T][C/A]TIGA[C/T]GA[A/ G]CTI- GA-3′). Products of the correct predicted size were analyzed by sequencing. Several clones encoded amino acids similar to those of S. cerevisiae ORC1. To identify a larger fragment, an internal exact primer (PO1PCR14, 5′- GGACCTTCTGTGGACTCACAAA-3′) was used in 3′ rapid amplification of cDNA ends (RACE) reactions containing 3′ anchor primer and 3′ adaptor primer (Gibco-BRL). Sequence analysis of amplified products revealed fragments with high homology to S. cerevisiae ORC1. One RACE product was used as a probe to screen a cDNA library derived from a human teratocarcinoma cell line [J. Skowronski, T. G. Fanning, M. F. Singer, Mol. Cell. Biol. 8, 1385 (1988)]. Positive plaques were purified and sequenced on both strands with the use of a semiautomated DNA sequencer (Applied Biosystems). The GenBank accession number is U40152. A comparison of the human cDNA against the National Center for Biotechnology Information (NCBI) databases was done with the BLAST algorithm [S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)].
35. Reverse transcription of human 293 total RNA was performed with degenerate primer PO1PCR7 (5′-[C/T]TCIGGIAG[A/G]TCCATIGT[A/G]TT-3′). The resulting cDNA was used as a template in PCR reactions containing primers PO1PCR7 and PO1PCR3 (5′-GT[C/G/T]CG[C/G/T][C/A]TIGA[C/T]GA[A/ G]CTI- GA-3′). Products of the correct predicted size were analyzed by sequencing. Several clones encoded amino acids similar to those of S. cerevisiae ORC1. To identify a larger fragment, an internal exact primer (PO1PCR14, 5′- GGACCTTCTGTGGACTCACAAA-3′) was used in 3′ rapid amplification of cDNA ends (RACE) reactions containing 3′ anchor primer and 3′ adaptor primer (Gibco-BRL). Sequence analysis of amplified products revealed fragments with high homology to S. cerevisiae ORC1. One RACE product was used as a probe to screen a cDNA library derived from a human teratocarcinoma cell line [J. Skowronski, T. G. Fanning, M. F. Singer, Mol. Cell. Biol. 8, 1385 (1988)]. Positive plaques were purified and sequenced on both strands with the use of a semiautomated DNA sequencer (Applied Biosystems). The GenBank accession number is U40152. A comparison of the human cDNA against the National Center for Biotechnology Information (NCBI) databases was done with the BLAST algorithm [S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)].
36. 90, tetrads from these diploids were dissected and found to segregate into two viable and two nonviable spores.
37. K. Gavin, M. Hidaka, B. Stillman, unpublished data.
38. 2-terminus is MGSYPYDYAHHHHHHG-SPRRKSLR . . . (33). The resulting plasmid (pKG14) was digested with Not I, blunt-ended with Klenow polymerase, then digested with Nde I. The vector pREP1 was digested with Sma I and Nde I and the tagged orc1 fragment was cloned into the Nde I-Sma I sites to make pKG15.
39. + ade6-210/ ade6-216 leu1-32/leu1-32 ura4-D18/ura4-D18) was transformed with pKG15 or pKG26 {a 6-kb Hind III genomic fragment in pWH5 isolated from an S. pombe genomic library [M. Caligiuri and D. Beach, Cell 72, 607 (1993)]} and sporulated on selective media. Colonies were streaked onto pombe minimal media containing adenine with or without thiamine.
40. K.A. Gavin and B. Stillman, unpublished data.
41. A 344-bp partial cDNA from A. thaliana encoding amino acids similar to those of a region of S. cerevisiae Orc2p was identified in the NCBI dbEST database (no. 1443). Primers based on the DNA sequence in the database (5′-CTTGCCCGGCTTTCTTCTTG-3′ and 5′-GTGTGAACAAGAAGAAAGC- CG-3′) were used to PCR-amplify a DNA fragment from an Arabidopsis cDNA library [D. Weigel, J. Alvarez, D. R. Smyth, M. F. Yanofsky, E. M. Meyerowitz, Cell 69, 843 (1992)]. Sequences flanking this on both sides were amplified with nested PCR primers complementary to each end of the fragment, and with a primer complementary to vector sequences. The primers complementary to the 5′ end are 5′-CACTCTTGAC-CATCGGAGTT-3′ and 5′-TGCTTCCGGCTCGTAT-GTTGTGTG-3′. The primers complementary to the 3′ end are 5′-CTTGCCCGGCTTTCTTCTTG-3′ and 5′-AGTCAAGTGACTTTAAA- CTC-3′. Finally, the 5′ end of the cDNA fragment was isolated by 5′-RACE with the use of two oligonucleotides complementary to the most 5′ end of the cDNAs (5′-AGCCACACCT-GAGCTCAAAGACCCACTTTG-3′ and 5′-GCCCC-ACCCAATTCTTTTGCCAAGAAGTAG-3′). The combined clones comprised the entire A. thaliana cDNA. The DNA sequence was determined for all fragments on both strands. The GenBank accession number is U40269.
42. A 446-bp partial cDNA from C. elegans encoding amino acids related to the S. cerevisiae Orc2p was identified in the NCBI dbEST database (no. 16625). A partial cDNA fragment was amplified by nested PCR with the use of DNA from a C. elegans λZAP cDNA library (gift of R. Barstead) and oligonucleotides complementary to the dbEST cDNA sequence (5′-ACGAGGGTGAAATTGATTGC-3′, 5′-TTGTGA-ATTGACGGCAAGAG-3′ and 5′-CTACAGTTGAT-CACATTTAC-3′). The amplified product was used as a probe to screen the λZAP cDNA library. A full-length cDNA was isolated and sequenced on both strands with the use of standard dideoxy sequencing. In addition, a genomic DNA fragment containing the partial cDNA sequence identified in the dbEST database was identified in the NCBI EMBL database (no. Z36949). Computer analysis of this DNA predicts an open reading frame encoding an Orc2-related protein consisting of nine exons spanning nucleotides 1596 to 5784. However, our results from the CeORC2 cDNA sequence revealed that the authentic protein begins at position 2618 and the first exon ends at 2793 and excludes the first five predicted exons. Before termination at 5784, the DNA from 5781 to 5882 is spliced out and the last exon terminates at 5998. The GenBank accession number is U40270.
43. A 340-bp partial cDNA encoding amino acids related to S. cerevisiae ORC2 was isolated by RT-PCR reaction using human HeLa cell mRNA. First-strand cDNA was synthesized from 2 μg of mRNA primed with oligo(dT). The resulting cDNA was used as a template for PCR with degenerate primers based on amino acids conserved among ScORC2, AtORC2, and CeORC2 (5′-[C/T]A[C/T]GGIGTIGGITCIAA[A/G][A/C]G-3′ and 5′-ATGTGGTCIA[C/T]IG[A/T]IGCIA[C/T]IA-3′). An amplified fragment was sequenced and found to be related to S. cerevisiae ORC2. This combination of primers also amplified a DNA fragment from K. lactis that was sequenced and found to be related to S. cerevisiae ORC2 (23). The human DNA fragment was used as a probe to isolate a full-length human cDNA [see (21)]. Positive clones were plaque-purified and sequenced on both strands. The GenBank accession number is U40268.
44. M. Gossen, D. T. S. Pak, S. Hansen, J. Acharya, M. Botchan, Science 270, 1674 (1995); A. E. Ehrenhofer-Murray, M. Gossen, D. T. S. Pak, M. R. Botchan, J. Rine, ibid., p. 167.
45. M. Gossen, D. T. S. Pak, S. Hansen, J. Acharya, M. Botchan, Science 270, 1674 (1995); A. E. Ehrenhofer-Murray, M. Gossen, D. T. S. Pak, M. R. Botchan, J. Rine, ibid., p. 167.
46. pKG28 contains the full-length cDNA encoding HsORC1 under the control of the cytomegalovirus promoter fused to a single copy of the T7 epitope in pCGT [A. Wilson, M. G. Peterson, W. Herr, Genes Dev. 9, 2445 (1995)]. Xba I and Bam HI sites were introduced into the cDNA encoding HsORC1 by PCR immediately upstream of the in-frame initiator methionine and immediately downstream of the stop codon, respectively. The entire coding region except for the 3′-most 120 nucleotides was replaced with a native DNA fragment, and sequencing of the 120 bp at the 3′ end revealed no mutations generated during PCR. Human 293 cells were transfected or mock-transfected with 10 μg of pKG28 by electroporation and were harvested 24 hours later. Whole-cell extracts were prepared and used for immunoprecipitation with rabbit antibody to amino acids 1 to 296 of HsOrc2p fused to GST. Immune complexes were collected on protein A-Sepharose (Pharmacia), washed, and separated by SDS-polyacrylamide gel electrophoresis (SOS-PAGE). The proteins were transferred to nitrocellulose, and immunoblot analysis was done with a monoclonal antibody to the T7 epitope (Novagen). The ECL system was used for detection (Amersham).
47. A. Kornberg and T. A. Baker, DNA Replication (Freeman, New York, 1991), vol. 2.
48. 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, He; 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.
49. We thank M. Hengartner, N. Kaplan, R. Martienssen, L. Rodgers, and J. Skowronski for providing various cDNA and genomic libraries; D. Casso and M. Caligiuri for S. pombe strains and vectors; A. Stenlund and M. Berg for help with transfections; M. Ockler for help with artwork; M. Weinreich and P. Kaufman for helpful discussions; and P. Kaufman for reading of the manuscript. Supported by the Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad to M.H. and by NIH grant CA13106 to B.S.