Analysis of DNA from Fired Cartridge Casings

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.

DNA Methylation Changes Caused by Lawsone

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.

Bioinformatic Analysis of Retroelement-Associated Sequences in Human and Mouse Promoters

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.

Study on the Derivatization Process Using N-O-bis-(trimethylsilyl)-trifluoroacetamide,N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide, Trimethylsilydiazomethane for the Determination of Fecal Sterols by Gas Chromatography-Mass Spectrometry

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.

Novel Inhibitor of E. coli DNA Adenine Methyltransferase (Ecodam)

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.

Molecular Analysis of Somaclonal Variation in Tissue Culture Derived Bananas Using MSAP and SSR Markers

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.

Study on the Derivatization Process Using N-O-bis-(trimethylsilyl)-trifluoroacetamide, N-(tert-butyldimethylsilyl)-N-methyltrifluoroace tamide, Trimethylsilydiazomethane for the Determination of Fecal Sterols by Gas Chromatography-Mass Spectrometry

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.