A Msel polymorphism in exon 48 of the dystrophin gene

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Office Forensic Science Service, Aldermaston, Berkshire. RG7 4PN, UK. I have sequenced a PCR-fragment from genomic DNA coding for the carboxy terminus ...
Nucleic Acids Research, Vol. 19, No. 20 5803

Human tyrosinase-like protein (TYRL) carboxy terminus: closer homology with the mouse protein than previously reported A.Urquhart Central Research and Support Establishment, Home Office Forensic Science Service, Aldermaston, Berkshire RG7 4PN, UK I have sequenced a PCR-fragment from genomic DNA coding for the carboxy terminus of tyrosinase-related protein (TYRL, gp75, Catalase B). 200 ng genomic DNA from whole blood or 10 ng from hair root sheath were amplified using primers TRP1 1 [5' TCGGGAGTTTAGTGTACCTGA 3'] and TRP12 [5' ATTCAACCAGGTGGTTTTGTG 3'], producing a 256 base pair fragment which was purified on a 3% agarose gel, excised and concentrated with Geneclean II (Bio 101, Inc.), before sequencing from both ends with the above primers using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.) and the Applied Biosystems 373A automated DNA sequencer. In 28 unrelated individuals of 5 racial origins (11 North European, 3 Middle Eastern, 7 Afro-Caribbean, 5 Indo-Pakistani, 2 Chinese), the only difference from the published sequence (1) was, in all cases, an apparent 4 base pair deletion (bases 1705-1708 in ref. 1). PCR and sequencing of cDNA from an S7 melanoma library (Clontech Labs, Inc.), the source of the published sequence (1), also showed the apparent deletion, so I suggest that the cDNA sequence reported in ref. 1 was that of an aberrant or unusual mRNA species. The lack of 4 base pairs lengthens the deduced amino acid sequence of TRP-I precursor by 10 residues to 537, the same length as the mouse protein (2) (see figure). Furthermore, 8 of the 12 carboxy terminus amino acids are conserved between the mouse and human, including a possible N-glycosylation site (Asn-Xaa-Ser) (3). The revised carboxy terminus sequence has been lodged in the EMBL database under Accession no. X60955. References: 1) Cohen,T., Muller,R.M., Tomita,Y. and Shibahara,S. (1990) Nucl. Acids Res. 18, 2807-2808. 2)

Shibahara,S., Tomita,Y., Sakakura,T., Nager,C., Chaudhuri,B. and Muller,R.M. (1986) Nucl. Acids Res. 14, 2413-2427. 3) Marshall,R.D. (1974) Biochem. Soc. Symp. 40, 17-26.

rmg 1710

1700

1720

1740

1750

(Cohen et al, 1990)

TGCTATGCTGAAGAAAGAATATGAAAAACTCCAGAATCCTAATCAGTCTGTGGTCTA... ...CysTyrAlaGluGluArgIleTER

Human TRP-I 3'terminus

...TGCTATGCTGAAGAATATGAAAAACTCCAGAATCCTAATCAGTCTGTGGTCTAACAA...

(this study)

...CysTyrAlaGluGluTyrGluLysLeuGlnAsnProAsnGlnSerValValTER

Mouse TRP-I 3 'terminus (Shibahara et al, 1986)

...

S7 TRP-I 3'terminus

...

A Msel polymorphism in exon 48 of the dystrophin gene S.C.Yau, R.G.Roberts, D.R.Bentley, C.G.Mathew and M.Bobrow Division of Medical and Molecular Genetics, Paediatric Research Unit, United Medical and Dental Schools, Guy's Hospital, London SE1 9RT, UK Source/Description: Two published nucleotide sequences for exon 48 of the dystrophin gene (1, 2) differ at a single base from the sequence of Koenig et al. (3). The discrepancy involves a C to A transversion at nucleotide position 7304 in the cDNA. Using amplification and mismatch detection (AMD) analysis (4) followed by direct sequencing, 7 out of 10 X chromosomes were shown to have the A nucleotide. The C to A base change creates a MseI restriction site which allows for rapid typing of the polymorphism by restriction digestion and electrophoresis of PCR amplified products (5) as shown by a study of 58 X chromosomes. PCR Primers: E48M F: 5' AAG CTT GAA GAC CTT GAA GAGC3' (from ref 3) E48M R: 5' CCT GAA TAA AGT CTT CCT TAC CAC AC 3' (from ref 1) Polymorphism: MseI digestion of the 249 bp PCR product yields two alleles: 108 bp (Al) and 85 bp plus 23 bp (A2), plus constant bands of 47, 30, 25, 21 and 18 bp. Frequency: Studied in 58 X chromosomes of unrelated individuals. PIC = 0.30 Frequency Allele (bp) AHI (108) 0.26 AH2 (85 + 23) 0.74 Chromosomal Location: Xp21.2. Mendelian Inheritance: Co-dominant X-linked segregation was observed in 4 families. Other Comments: PCR is performed using 30 cycles of 48 sec at 93°C, 48 sec at 60°C, 3 min at 72°C. MseI digested products are analysed on a 7% horizontal polyacrylamide minigel. This new polymorphism should prove useful in linkage studies in DMD families and particularly in determining carrier status for relatives of the 25 % of deletion cases that involve this exon (see

figure). Acknowledgements: This work was supported by the Medical Research Council, the Generation Trust, and the Muscular Dystrophy Group of Great Britain and Northern Ireland. References: 1) Chamberlain,J.S. et al. (1988) Nucl. Acids Res. 16, 11141-11156. 2) Rosenthal,A. et al. (1989) Nucl. Acids Res. 17, 5391. 3) Koenig,M. et al. (1988) Cell 53, 219-228. 4) Cotton,R.G.H. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 4397-4401. 5) Saiki,R.K. et al. (1988) Science 239, 487 -491.

CGCTATGCTGAGGACTATGAGGAGCTCCCGAATCCTAACCACTCCATGGTCTGATAG...

ArgTyrAlaGluAspTyrGluGluLeuProAsnProAsnHisSerMetValTER

...

Nucleotides

are

numbered as in reference 1.

A2

A1fA2

A1/A2

Al/A2

Al

Deleted

-A l(l 08bp)

-A2(8 5bp) -Constant Bands