Simple quantitative PCR approach to reveal naturally occurring and mutation-induced repetitive sequence variation on the Drosophila Y chromosome.

Keith A Maggert,Nadia Singh,John C Aldrich

doi:10.1371/journal.pone.0109906

Abstract

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.

Highlights

A significant portion of most eukaryotic genomes is composed of repetitive DNA elements [1]
Design of Real-Time-Based Quantitative PCR Approach Large blocks of simple pentameric repeats AACAC and AAGAC are constituents of the Drosophila Y chromosome [7,41], accounting for less than about 2% and about 20%, respectively, of the Y; the remaining balance largely resides in the pericentric heterochromatin of chromosome 2
A number of methods currently exist for determining the copy number of satellite DNAs –the repetitive simple sequences that comprise nearly half of most eukaryotic genomes

Summary

Introduction

A significant portion of most eukaryotic genomes is composed of repetitive DNA elements [1]. Highly-repetitive heterochromatic satellite sequences (e.g., AAGAG, AATAT, AAGAGAG) house a variety of biological phenomena including centromere function, chromosome cohesion and pairing, nuclear organization, control of recombination, species-compatibilities, replication rate, and gene regulatory variation [9,10,11,12,13,14], understanding their function mechanistically has lagged far behind sophisticated understanding of the function of euchromatic sequences. This is due in large part to the difficulty in handling these sequences with modern molecular biological approaches. Next-generation sequencing technology has increased the rate with which we have learned about the structure and variation of euchromatin, but the heterochromatic portion of the genome remains relatively ignored in its characterization [3,15], even very recently not rising to the level of notice in debate over the role of ‘‘junk’’DNA [16,17]

Methods

Results

Conclusion