Rearranging the centromere of the human Y chromosome with fC31 integrase

S Malla, F Dafhnis-Calas, J F Y Brookfield, Maggie Smith, W R A Brown

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We have investigated the ability of the integrase from the Streptomyces phi C31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phi C31 integrase attB site and the chicken genome.
Original languageEnglish
Pages (from-to)6101-6113
Number of pages13
JournalNucleic Acids Research
Volume33
Issue number19
DOIs
Publication statusPublished - 2005

Keywords

  • site-specific integration
  • mammalian-cells
  • human minichromosomes
  • synaptic complex
  • Cre recombinase
  • mosaic analysis
  • DNA
  • architecture
  • selection
  • vectors

Cite this

Malla, S., Dafhnis-Calas, F., Brookfield, J. F. Y., Smith, M., & Brown, W. R. A. (2005). Rearranging the centromere of the human Y chromosome with fC31 integrase. Nucleic Acids Research, 33(19), 6101-6113. https://doi.org/10.1093/nar/gki922

Rearranging the centromere of the human Y chromosome with fC31 integrase. / Malla, S; Dafhnis-Calas, F; Brookfield, J F Y; Smith, Maggie; Brown, W R A .

In: Nucleic Acids Research, Vol. 33, No. 19, 2005, p. 6101-6113.

Research output: Contribution to journalArticle

Malla, S, Dafhnis-Calas, F, Brookfield, JFY, Smith, M & Brown, WRA 2005, 'Rearranging the centromere of the human Y chromosome with fC31 integrase', Nucleic Acids Research, vol. 33, no. 19, pp. 6101-6113. https://doi.org/10.1093/nar/gki922
Malla S, Dafhnis-Calas F, Brookfield JFY, Smith M, Brown WRA. Rearranging the centromere of the human Y chromosome with fC31 integrase. Nucleic Acids Research. 2005;33(19):6101-6113. https://doi.org/10.1093/nar/gki922
Malla, S ; Dafhnis-Calas, F ; Brookfield, J F Y ; Smith, Maggie ; Brown, W R A . / Rearranging the centromere of the human Y chromosome with fC31 integrase. In: Nucleic Acids Research. 2005 ; Vol. 33, No. 19. pp. 6101-6113.
@article{6ec9e5a047374ca4b155758bc6dfe77d,
title = "Rearranging the centromere of the human Y chromosome with fC31 integrase",
abstract = "We have investigated the ability of the integrase from the Streptomyces phi C31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54{\%} of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phi C31 integrase attB site and the chicken genome.",
keywords = "site-specific integration, mammalian-cells, human minichromosomes, synaptic complex, Cre recombinase, mosaic analysis, DNA, architecture, selection, vectors",
author = "S Malla and F Dafhnis-Calas and Brookfield, {J F Y} and Maggie Smith and Brown, {W R A}",
year = "2005",
doi = "10.1093/nar/gki922",
language = "English",
volume = "33",
pages = "6101--6113",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "19",

}

TY - JOUR

T1 - Rearranging the centromere of the human Y chromosome with fC31 integrase

AU - Malla, S

AU - Dafhnis-Calas, F

AU - Brookfield, J F Y

AU - Smith, Maggie

AU - Brown, W R A

PY - 2005

Y1 - 2005

N2 - We have investigated the ability of the integrase from the Streptomyces phi C31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phi C31 integrase attB site and the chicken genome.

AB - We have investigated the ability of the integrase from the Streptomyces phi C31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phi C31 integrase attB site and the chicken genome.

KW - site-specific integration

KW - mammalian-cells

KW - human minichromosomes

KW - synaptic complex

KW - Cre recombinase

KW - mosaic analysis

KW - DNA

KW - architecture

KW - selection

KW - vectors

U2 - 10.1093/nar/gki922

DO - 10.1093/nar/gki922

M3 - Article

VL - 33

SP - 6101

EP - 6113

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 19

ER -