hRAD30 mutations in the variant form of xeroderma pigmentosum

Science

Volumn 285, Issue 5425, 1999, Pages 263-265

  Johnson, Robert E ^a   Kondratick, Christine M ^a   Prakash, Satya ^a   Prakash, Louise ^a  

^a UNIVERSITY OF TEXAS MEDICAL BRANCH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; AUTOSOMAL RECESSIVE DISORDER; FRAMESHIFT MUTATION; GENE MUTATION; HUMAN; NONHUMAN; PRIORITY JOURNAL; SKIN CANCER; SKIN SENSITIVITY; SUN EXPOSURE; XERODERMA PIGMENTOSUM;

ALLELES; AMINO ACID SEQUENCE; CELL LINE; DNA DAMAGE; DNA REPAIR; DNA REPLICATION; DNA, COMPLEMENTARY; DNA-DIRECTED DNA POLYMERASE; FRAMESHIFT MUTATION; HUMANS; MOLECULAR SEQUENCE DATA; MUTATION; NEOPLASMS, RADIATION-INDUCED; PROTEIN BIOSYNTHESIS; PYRIMIDINE DIMERS; SACCHAROMYCES CEREVISIAE; SEQUENCE ALIGNMENT; SEQUENCE DELETION; SKIN NEOPLASMS; ULTRAVIOLET RAYS; XERODERMA PIGMENTOSUM;

EID: 0033538470     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5425.263     Document Type: Article

References (25)

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17. The hRAD30 done was derived from a high-throughput cDNA screening service (Genome Systems, St. Louis, MO). Oligonucleotides N4920 (5′-CCCGGTACTTGGT-GAGGTTAGCTTTCCCACGGC-3′) and N4921 (5′-ATA-ATTGCAGTGAGTTATGAAGCTCGCATTTGGAG-3′), which amplify nucleotides +139 to +280 of the hRAD30 gene, were derived from the H96386 cDNA sequence and used to generate a 142-bp PCR fragment. This fragment was used as a probe to screen a human spleen cDNA library. One done, 21749, was found to carry a 3.0-kb insert in vector pcDNA2.1 (Invitrogen).
18. RNA was isolated from various cell lines by use of the Tri-Reagent protocol from Molecular Research Center (Cincinnati, OH). hRad30 cDNA was generated from total human RNA by use of the Superscript One Step reverse transcriptase (RT)-PCR system (Gibco-BRL) and amplified in three overlapping fragments. Fragment 1 was amplified with oligonucleotides RT5′ (5′-GG-GAATAAATCTCGCTCGAAACTCACTGGACCGG-3′) and N4920 (9); this fragment encompasses nt -152 to +280 of hRAD30. Fragment 2 was generated with oligonucleotides N4921 (9) and N4918 (5′-CTTTTGC-CTTGATGAGATACGGCAGAAACAACCAGGG-3′); this fragment encompasses nt +139 to +2048 of hRAD30. Fragment 3 encompasses nt +1738 to +2267 and was amplified with oligonudeotides N4919 (5′-GGGGT-GTCGAAGCTAGAAGAATCCTCTAAAGCAACTCC-3′) and RT3′ (5′-TTATTTTTTGTATTAAAATTTCAT-AATTCCCTTTCTCAG-3′). Amplified cDNAs from cell lines and done 21749 (9) were sequenced with the Thermo Sequenase kit (Amersham Pharmacia Biotech). In addition to the oligonudeotides used for RT-PCR, we used the following oligonucleotides as primers to sequence the hMD30 gene: 5′ Seq (5′-AACTTCTTAG-CATCATCTGCCCAC-3′), N5035 (5′-GCCTATC-TCGGCAGACTTGTTGCC-3′), N5321 (5′-GAAAT-GAGAGCAGCCATAGAGAGGG-3′), N5086 (5′-GA-GATCCTAGGGATAGAATACATGGG-3′), N5322 (5′-GAGCATCATAGCGGGTAAGGGC-3′), N5087 (5′-CTGGCACCTTTGGCAGAGAACTTGGG-3′), 1565AS (5′-CTCAGTTCCTGTACTTTGACTCG-3′), N5036 (5′-GGGCCAAATCCATCTGCAGG-3′), and 3′ Seq (5′-GCAGAAATCCTTTTTGCAGCCCC-3′). Samples were run on 6 to 8% tris-taurine-EDTA polyacrylamide gels containing 8 M urea. Gels were fixed in 10% acetic acid, 10% methanol solution and dried before autoradiography. Each of the mutations in the XP-V cell lines were independently amplified and sequenced three or four times.
19. Supplemental information is available on Science Online at www.sciencemag.org/feature/data/1041880. shl.
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21. A. A. Rousch, M. Suarez, E. C. Friedberg, M. Radman, W. Siede, Mol. Gen. Genet. 257, 686 (1998).
22. Cell lines were obtained from the Coriell Cell Line Repository (CCR, Camden, NJ). Lymphoblast cell lines were normal (GM00892B), XPA (GM02250D), XPB (GM02252A), XPC (GM02246C), and XPPHBE (GM02449C). Fibroblast cell lines were XP1CH (GM03055), XP2CH (GM03053), XP115LO (GM02359A), XP30RO (GM03617), XP5MA (GM03379), XP60U (GM03518), and XP1SF (GM06090).
23. To quantitate the mutant and wild-type hRad30 transcripts in the XP-V cell lines XPPHBE (GM02449C) and XP15F (GM06090), we cloned the amplified cDNAs encompassing the respective mutations from each cell line into pUC19-based vectors and sequenced 20 independent clones from each cell line.
24. C. M. Kondratick, M. T. Washington, R. E. Johnson, S. Prakash, L. Prakash, unpublished observations.
25. Supported by NIH grant GM19261.