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Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Shanks ME, May CA, Dubrova YE, Balaresque P, Rosser ZH, Adams SM, Jobling MA - Mutat. Res. (2008)

Bottom Line: Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias.Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant).There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, UK.

ABSTRACT
Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

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Repeat type, structure of progenitor MSY1 array, and junction primer strategy for mapping mutants. In the middle is shown a schematic structure of the donor allele, with repeat units indicated by circles and sequences given in the key. Arrows indicate primers. Below and above are shown electropherograms showing, respectively the results of typing the 1, 3 and 3, 4 repeat unit boundaries, using primer combinations Y1A+/JUN-1, 3 and Y1B+/JUN-3, 4. Junction primers are fluorescently labelled (‘F’). RFU: relative fluorescent units. The junction primers are directed at the boundaries, but also yield PCR products corresponding to other local repeats through mispriming. The putative hairpin adopted by a type 4 repeat is also shown.
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fig1: Repeat type, structure of progenitor MSY1 array, and junction primer strategy for mapping mutants. In the middle is shown a schematic structure of the donor allele, with repeat units indicated by circles and sequences given in the key. Arrows indicate primers. Below and above are shown electropherograms showing, respectively the results of typing the 1, 3 and 3, 4 repeat unit boundaries, using primer combinations Y1A+/JUN-1, 3 and Y1B+/JUN-3, 4. Junction primers are fluorescently labelled (‘F’). RFU: relative fluorescent units. The junction primers are directed at the boundaries, but also yield PCR products corresponding to other local repeats through mispriming. The putative hairpin adopted by a type 4 repeat is also shown.

Mentions: The progenitor array structure was determined using a radioactive MVR-PCR technique [12]. Internal structures of all single-molecule products were defined using primers targeted at the junctions between blocks of repeat types [19] (Fig. 1), paired with flanking primers 5′-labelled with 6-FAM. Primer JUN-1,3F (5′-CGC TGC CAA CTA CCG CAC ATG TAT ACA TGA TGT ATA TTG TGT ATA ATA TAC ATC ATG TAT ATT G-3′) was specific to the type 1/type 3 junction, and paired with Y1A+; and primer JUN-3,4R (5′-CGC TGC CAA CTA CCG CAC ATG CAC AAT ATA CAT CAT GTA TAT TAT ACA TAA TAT ACA TC-3′) was specific to the type 3/type 4 junction, and paired with Y1B+. Reactions contained Amplitaq Gold buffer (Applied Biosystems), 1.5 mM MgCl2, 1 μg/ml BSA (NEBL), 0.2 mM dNTPs, 0.04U Amplitaq Gold (Applied Biosystems), and 1 μM each primer, together with 1 μl primary PCR product. General PCR conditions were: 95 °C for 11 min, followed by 95 °C for 1 min, 65 °C for 3.5 min and 72 °C for 5 min for 35 cycles. Products were resolved on an ABI3100 Genetic Analyzer and sizes determined with reference to a ROX-400 standard (Applied Biosystems).


Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Shanks ME, May CA, Dubrova YE, Balaresque P, Rosser ZH, Adams SM, Jobling MA - Mutat. Res. (2008)

Repeat type, structure of progenitor MSY1 array, and junction primer strategy for mapping mutants. In the middle is shown a schematic structure of the donor allele, with repeat units indicated by circles and sequences given in the key. Arrows indicate primers. Below and above are shown electropherograms showing, respectively the results of typing the 1, 3 and 3, 4 repeat unit boundaries, using primer combinations Y1A+/JUN-1, 3 and Y1B+/JUN-3, 4. Junction primers are fluorescently labelled (‘F’). RFU: relative fluorescent units. The junction primers are directed at the boundaries, but also yield PCR products corresponding to other local repeats through mispriming. The putative hairpin adopted by a type 4 repeat is also shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2599865&req=5

fig1: Repeat type, structure of progenitor MSY1 array, and junction primer strategy for mapping mutants. In the middle is shown a schematic structure of the donor allele, with repeat units indicated by circles and sequences given in the key. Arrows indicate primers. Below and above are shown electropherograms showing, respectively the results of typing the 1, 3 and 3, 4 repeat unit boundaries, using primer combinations Y1A+/JUN-1, 3 and Y1B+/JUN-3, 4. Junction primers are fluorescently labelled (‘F’). RFU: relative fluorescent units. The junction primers are directed at the boundaries, but also yield PCR products corresponding to other local repeats through mispriming. The putative hairpin adopted by a type 4 repeat is also shown.
Mentions: The progenitor array structure was determined using a radioactive MVR-PCR technique [12]. Internal structures of all single-molecule products were defined using primers targeted at the junctions between blocks of repeat types [19] (Fig. 1), paired with flanking primers 5′-labelled with 6-FAM. Primer JUN-1,3F (5′-CGC TGC CAA CTA CCG CAC ATG TAT ACA TGA TGT ATA TTG TGT ATA ATA TAC ATC ATG TAT ATT G-3′) was specific to the type 1/type 3 junction, and paired with Y1A+; and primer JUN-3,4R (5′-CGC TGC CAA CTA CCG CAC ATG CAC AAT ATA CAT CAT GTA TAT TAT ACA TAA TAT ACA TC-3′) was specific to the type 3/type 4 junction, and paired with Y1B+. Reactions contained Amplitaq Gold buffer (Applied Biosystems), 1.5 mM MgCl2, 1 μg/ml BSA (NEBL), 0.2 mM dNTPs, 0.04U Amplitaq Gold (Applied Biosystems), and 1 μM each primer, together with 1 μl primary PCR product. General PCR conditions were: 95 °C for 11 min, followed by 95 °C for 1 min, 65 °C for 3.5 min and 72 °C for 5 min for 35 cycles. Products were resolved on an ABI3100 Genetic Analyzer and sizes determined with reference to a ROX-400 standard (Applied Biosystems).

Bottom Line: Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias.Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant).There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, UK.

ABSTRACT
Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

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