Observing the darkest matter of the genome : expression of human endogenous retrovirus W elements
Author: Nellåker, Christoffer
Date: 2008-05-30
Location: Hillarpsalen, Retzius väg 8, Karolinska Institutet
Time: 09.30
Department: Institutionen för neurovetenskap / Department of Neuroscience
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thesis.pdf (1.854Mb)
Abstract
The human genome is composed of coding genes and vast stretches of
sequences largely considered junk. Researchers are, however, uncovering
widespread and extensive transcription of not only the coding, but also
of the non-coding sequences in the genomes of many species. Transcripts
that do not code for any protein are thought to carry out their potential
functions by directly interacting with other sequences and proteins by
their base-pairing capabilities or secondary structures. Since little is
known about non-coding DNA and their RNA transcripts, they have been
called the dark matter of the genome. Half the human genome is composed
of repetitive sequences, about eight percent by ancient remnants of
retroviral infections called human endogenous retroviruses (HERV). These
repetitive elements are usually excluded from most studies of expressed
sequences as they are methodologically problematic to identify
unambiguously. The dogma has been that degenerated viral sequences are junk and are for the most part transcriptionally silent. This is being revised because of observation of transcription of these elements in human tissues and expression variations associated to human diseases. These repetitive regions could be called the darkest matter of the
genome.
In this thesis are included observations of expression patterns of HERV elements and increased expression and alterations associated to exogenous virus infections. An evaluation of the currently available sequence specific assays and a novel melting temperature (Tm) analysis method for studying expression patterns of highly repetitive and homologous sequences is presented herein. The Tm analysis method was further developed with: i) the use of a temperature probe to normalize for temperature deviations in the thermocycler instrument, ii) a curve fit algorithm to interpolate exact temperatures from multiple data points and iii) a new approach to analyzing obtained Tm with mixture models for an impartial and objective statistical analysis. Using these methods, we studied the expression patterns of individual elements within one HERV family in human tissues. We found significant differences between expression patterns of HERV between human tissues and between individuals to an extent similar to that which would be expected for coding transcripts. The observations and methods developed in the course of this thesis might hopefully help in casting some light on the expression, regulation and functions of these RNAs containing highly repetitive sequences.
In this thesis are included observations of expression patterns of HERV elements and increased expression and alterations associated to exogenous virus infections. An evaluation of the currently available sequence specific assays and a novel melting temperature (Tm) analysis method for studying expression patterns of highly repetitive and homologous sequences is presented herein. The Tm analysis method was further developed with: i) the use of a temperature probe to normalize for temperature deviations in the thermocycler instrument, ii) a curve fit algorithm to interpolate exact temperatures from multiple data points and iii) a new approach to analyzing obtained Tm with mixture models for an impartial and objective statistical analysis. Using these methods, we studied the expression patterns of individual elements within one HERV family in human tissues. We found significant differences between expression patterns of HERV between human tissues and between individuals to an extent similar to that which would be expected for coding transcripts. The observations and methods developed in the course of this thesis might hopefully help in casting some light on the expression, regulation and functions of these RNAs containing highly repetitive sequences.
List of papers:
I. Nellåker C, Yao Y, Jones-Brando L, Mallet F, Yolken RH, Karlsson H (2006). "Transactivation of elements in the human endogenous retrovirus W family by viral infection." Retrovirology 3: 44
Pubmed
II. Yao Y, Nellåker C, Karlsson H (2006). "Evaluation of minor groove binding probe and Taqman probe PCR assays: Influence of mismatches and template complexity on quantification." Mol Cell Probes 20(5): 311-6. Epub 2006 Apr 21
Pubmed
III. Nellåker C, Wållgren U, Karlsson H (2007). "Molecular beacon-based temperature control and automated analyses for improved resolution of melting temperature analysis using SYBR I green chemistry." Clin Chem 53(1): 98-103. Epub 2006 Nov 16
Pubmed
IV. Nellåker C, Uhrzander F, Tyrcha J, Karlsson H (2008). "Mixture models for analysis of melting temperature data." (Submitted)
V. Nellåker C, Uhrzander F, Tyrcha J, Karlsson H (2008). "Expression of transcripts containing human endogenous retrovirus W elements in human tissues." (Submitted)
I. Nellåker C, Yao Y, Jones-Brando L, Mallet F, Yolken RH, Karlsson H (2006). "Transactivation of elements in the human endogenous retrovirus W family by viral infection." Retrovirology 3: 44
Pubmed
II. Yao Y, Nellåker C, Karlsson H (2006). "Evaluation of minor groove binding probe and Taqman probe PCR assays: Influence of mismatches and template complexity on quantification." Mol Cell Probes 20(5): 311-6. Epub 2006 Apr 21
Pubmed
III. Nellåker C, Wållgren U, Karlsson H (2007). "Molecular beacon-based temperature control and automated analyses for improved resolution of melting temperature analysis using SYBR I green chemistry." Clin Chem 53(1): 98-103. Epub 2006 Nov 16
Pubmed
IV. Nellåker C, Uhrzander F, Tyrcha J, Karlsson H (2008). "Mixture models for analysis of melting temperature data." (Submitted)
V. Nellåker C, Uhrzander F, Tyrcha J, Karlsson H (2008). "Expression of transcripts containing human endogenous retrovirus W elements in human tissues." (Submitted)
Issue date: 2008-05-09
Rights:
Publication year: 2008
ISBN: 978-91-7357-588-1
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