Abstract: DNA analysis has been widely accepted as providing
valuable evidence concerning the identity of the source of biological
traces. Our work has showed that DNA samples can survive on
cartridges even after firing. The study also raised the possibility of
determining other information such as the age of the donor. Such
information may be invaluable in certain cases where spent cartridges
from automatic weapons are left behind at the scene of a crime. In
spite of the nature of touch evidence and exposure to high chamber
temperatures during shooting, we were still capable to retrieve
enough DNA for profile typing. In order to estimate age of
contributor, DNA methylation levels were analyzed using EpiTect
system for retrieved DNA. However, results were not conclusive, due
to low amount of input DNA.
Abstract: Lawsone is a pigment that occurs naturally in plants.
It has been used as a skin and hair dye for a long time. Moreover, its
different biological activities have been reported. The present study
focused on the effect of lawsone on a plant cell model represented by
tobacco BY-2 cell suspension culture, which is used as a model
comparable with the HeLa cells. It has been shown that lawsone
inhibits the cell growth in the concentration-dependent manner. In
addition, changes in DNA methylation level have been determined.
We observed decreasing level of DNA methylation in the presence of
increasing concentrations of lawsone. These results were
accompanied with overproduction of reactive oxygen species (ROS).
Since epigenetic modifications can be caused by different stress
factors, there could be a connection between the changes in the level
of DNA methylation and ROS production caused by lawsone.
Abstract: Mammalian genomes contain large number of
retroelements (SINEs, LINEs and LTRs) which could affect
expression of protein coding genes through associated transcription
factor binding sites (TFBS). Activity of the retroelement-associated
TFBS in many genes is confirmed experimentally but their global
functional impact remains unclear. Human SINEs (Alu repeats) and
mouse SINEs (B1 and B2 repeats) are known to be clustered in GCrich
gene rich genome segments consistent with the view that they
can contribute to regulation of gene expression. We have shown
earlier that Alu are involved in formation of cis-regulatory modules
(clusters of TFBS) in human promoters, and other authors reported
that Alu located near promoter CpG islands have an increased
frequency of CpG dinucleotides suggesting that these Alu are
undermethylated. Human Alu and mouse B1/B2 elements have an
internal bipartite promoter for RNA polymerase III containing
conserved sequence motif called B-box which can bind basal
transcription complex TFIIIC. It has been recently shown that TFIIIC
binding to B-box leads to formation of a boundary which limits
spread of repressive chromatin modifications in S. pombe. SINEassociated
B-boxes may have similar function but conservation of
TFIIIC binding sites in SINEs located near mammalian promoters
has not been studied earlier. Here we analysed abundance and
distribution of retroelements (SINEs, LINEs and LTRs) in annotated
sequences of the Database of mammalian transcription start sites
(DBTSS). Fractions of SINEs in human and mouse promoters are
slightly lower than in all genome but >40% of human and mouse
promoters contain Alu or B1/B2 elements within -1000 to +200 bp
interval relative to transcription start site (TSS). Most of these SINEs
is associated with distal segments of promoters (-1000 to -200 bp
relative to TSS) indicating that their insertion at distances >200 bp
upstream of TSS is tolerated during evolution. Distribution of SINEs
in promoters correlates negatively with the distribution of CpG
sequences. Using analysis of abundance of 12-mer motifs from the
B1 and Alu consensus sequences in genome and DBTSS it has been
confirmed that some subsegments of Alu and B1 elements are poorly
conserved which depends in part on the presence of CpG
dinucleotides. One of these CpG-containing subsegments in B1
elements overlaps with SINE-associated B-box and it shows better
conservation in DBTSS compared to genomic sequences. It has been
also studied conservation in DBTSS and genome of the B-box
containing segments of old (AluJ, AluS) and young (AluY) Alu
repeats and found that CpG sequence of the B-box of old Alu is
better conserved in DBTSS than in genome. This indicates that Bbox-
associated CpGs in promoters are better protected from
methylation and mutation than B-box-associated CpGs in genomic
SINEs. These results are consistent with the view that potential
TFIIIC binding motifs in SINEs associated with human and mouse
promoters may be functionally important. These motifs may protect
promoters from repressive histone modifications which spread from
adjacent sequences. This can potentially explain well known
clustering of SINEs in GC-rich gene rich genome compartments and
existence of unmethylated CpG islands.
Abstract: Fecal sterol has been proposed as a chemical indicator
of human fecal pollution even when fecal coliform populations have
diminished due to water chlorination or toxic effects of industrial
effluents. This paper describes an improved derivatization procedure
for simultaneous determination of four fecal sterols including
coprostanol, epicholestanol, cholesterol and cholestanol using gas
chromatography-mass spectrometry (GC-MS), via optimization study
on silylation procedures using N-O-bis
(trimethylsilyl)-trifluoroacetamide (BSTFA), and
N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide
(MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and
tert-butyldimethylsilyl (TBS) derivatives, respectively. Two
derivatization processes of injection-port derivatization and water bath
derivatization (60 oC, 1h) were inspected and compared. Furthermore,
the methylation procedure at 25 oC for 2h with
trimethylsilydiazomethane (TMSD) for fecal sterols analysis was also
studied. It was found that most of TMS derivatives demonstrated the
highest sensitivities, followed by methylated derivatives. For BSTFA
or MTBSTFA derivatization processes, the simple injection-port
derivatization process could achieve the same efficiency as that in the
tedious water bath derivatization procedure.
Abstract: EcoDam is an adenine-N6 DNA methyltransferase
that methylates the GATC sites in the Escherichia coli genome.
DNA-adenine methylation is not present in higher eukaryotes
including humans. These observations raise the possibility that dam
inhibitors may be used as anti-microbial agents. Polyphosphate
(Poly(P)) is an important metabolite and signaling molecule in
prokaryotes and eukaryotes. Here, by using gel retardation
experiments to investigate the competition of DNA binding by
EcoDam in the presence of polyphosphate, we found that Poly (P)
strongly interferes with DNA binding by EcoDam, while same
concentration of monophosphate does not. In addition, we
demonstrated that Poly (P) binding inhibits the activity of EcoDam
and our results suggest that Poly (P) led to strong inhibition of the
EcoDam catalytic activity, while monophosphate had only moderate
effect.
Abstract: The project was undertaken to determine the effects of modified tissue culture protocols e.g. age of culture and hormone levels (2,4-D) in generating somaclonal variation. Moreover, the utility of molecular markers (SSR and MSAP) in sorting off types/somaclones were investigated.
Results show that somaclonal variation is in effect due to prolonged subculture and high 2,4-D concentration. The resultant variation was observed to be due to high level of methylation events specifically cytosine methylation either at the internal or external cytosine and was identified by methylation sensitive amplification polymorphism (MSAP).Simple sequence repeats (SSR) on the other hand, was able to associate a marker to a trait of interest.
These therefore, show that molecular markers can be an important tool in sorting out variation/mutants at an early stage.
Abstract: Fecal sterol has been proposed as a chemical indicator
of human fecal pollution even when fecal coliform populations have
diminished due to water chlorination or toxic effects of industrial
effluents. This paper describes an improved derivatization procedure
for simultaneous determination of four fecal sterols including
coprostanol, epicholestanol, cholesterol and cholestanol using gas
chromatography-mass spectrometry (GC-MS), via optimization study
on silylation procedures using N-O-bis
(trimethylsilyl)-trifluoroacetamide (BSTFA), and
N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide
(MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and
tert-butyldimethylsilyl (TBS) derivatives, respectively. Two
derivatization processes of injection-port derivatization and water bath
derivatization (60 oC, 1h) were inspected and compared. Furthermore,
the methylation procedure at 25 oC for 2h with
trimethylsilydiazomethane (TMSD) for fecal sterols analysis was also
studied. It was found that most of TMS derivatives demonstrated the
highest sensitivities, followed by methylated derivatives. For BSTFA
or MTBSTFA derivatization processes, the simple injection-port
derivatization process could achieve the same efficiency as that in the
tedious water bath derivatization procedure.