Functional Characterization of Yiro10w (DSN1) and Yor228c Genes in Saccharomyces Cerevisiae

The aim of this project was to functionally decipher two novel genes of Saccharomyces cerevisiae (YOR228C and YIROIOW). Four levels of study were undertaken to achieve the aims of this study including bioinformatics data mining and prediction, gene deletion study, phenotypic analysis and protein profiling. Sequence analysis of YOR228C predicted the expression of a 34 kDa transmembrane protein of low abundance with the potential of forming a homodimer. It could be a stress-responsive gene that also regulates cell growth in yeast but it was not an essential component of the cell. On the other hand, Yir010wp has a size of 66 kDa and would be a heterodimer in a complex. This low abundant protein may be related to cell division cycle as it was found to be incorporated within the yeast spindle pole body. The necessity for its exact stoichiometry was a sign of gene dosage sensitivity as demonstrated by the reduction in heterologous fitness. Deletion of a single copy of YIROIOW caused chromosomal segregation error leading to aneuploidy. Furthermore, double knockout of the gene from the genome was lethal, implying its essentiality to the yeast cell. As a whole, this study has successfully elucidated the general functions of the two genes under investigation, namely the relationship between YOR228C to growthlstressresponse and the link between YIROIOW to the cell division cycle

Electrophoresis : what does a century old technology hold for the future of separation science?

Electrophoretic separation was first demonstrated in the year of 1807 and has since been a staple tool used by biologists and chemists for more than a century since its inception. From the initial crude paper electrophoresis system to today’s modern automated electrophoresis system, the development of electrophoresis systems have been driven by the advancement of technology such as miniaturization, precision engineering, biochemistry, electrical and electronics. These advancements were introduced to meet the requirement for faster and better resolution of results. This paper reviews the evolution of the electrophoresis technology over one century and provides an insight into the possible future development of electrophoresis.Various aspects of the electrophoresis system such as the performances, designs, usages, separation phases, and biochemistry were analyzed. The technological advancements for this field have been evidenced by the increasing complexity of the electrophoresis system. A peek into the possible future for the world of electrophoresis has been provided by drawing insights from the missing links of current technologies. It is both exciting and equally perplexing to explore the promises that this seeming simple separation technology holds for the future

Saccharomyces cerevisiae kinetochore protein (rDsn1p) induced apoptosis in Chinese hamster ovary cells

Dsn1p is a member of the MIND complex that forms part of the yeast kinetochore, which is essential for the proper chromosomal segregation during cell division. Its functionality is gene dosage dependent and it has characteristics of haploinsufficiency. Bioinformatics alignments predicted the existence of nuclear homologues in higher eukaryotic organisms. Literature on the possibility of Dsn1p being a functional homologue of these organisms is scarce. In this study we employed recombinant DNA expression technology to explore whether Dsn1p can function in a mammalian cell line, Chinese Hamster Ovary (CHO). Expression of rDsn1p in CHO cells induced cytopathic effects including changes in cellular morphology and cell size. Inhibition of cell growth was observed at the beginning the fourth post-transfection week. The recombinant CHO cell culture showed cytotoxic effects following the accumulation of the Dsn1p, resulting in apoptotic cell death; as evidenced by the presence of nuclear fragmentation and surface blebbing in the dying cells. This suggests that rDsn1p may interact with the counterpart/ligand of the nuclear homologue of this protein in CHO cells, resulting in nuclear anomalies and inhibition of cell growth, as observed in our previous study using yeast cells

A low cost, simplified and battery-powered mobile electrophoresis system suitable as edutainment tool.

Electrophoresis originated since 1807 and has been a staple tool used by biologist and chemist over the centuries since its inception. From paper electrophoresis system to today’s modern automated electrophoresis system, the development of electrophoresis systems have been driven by the advancement of technology and also by the requirement of better and faster resolution of results. This paper reviews the progress of electrophoresis over the decades and into possible future development of electrophoresis

Extra dose of Dsn1p (Yir010wp) expressed from episomal plasmid in normal Saccharomyces cerevisiae diploid wild type has augmented growth rate and altered budding pattern, suggesting its growth stimulating property

DSN1 is a spindle pole body component that functions in the chromosomal segregation process of yeast. The effect of additional DSN1 gene dosage on the yeast cell has yet to be established. The work from this study indicated that an extra gene dosage of DSN1 caused abnormalities in both cellular growth and budding pattern. The yeast cells carrying an additional copy of DSN1 gene have an increased growth rate and they formed a highly retarded multiple bud complex. These phenomena suggest possible growth stimulating property of the gene product

Genomic DNA extraction from medicinal plants available in Malaysia using a TriOmicTM improved extraction kit

DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmicTM extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1plus buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmicTM extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete

Cloning of a novel phytase from an anaerobic rumen bacterium, Mitsuokella jalaludinii, and its expression in Escherichia coli

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimized and characterized. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation

A process and kit for isolation and purifying biology materials.

The present invention relates to a process and kit useful for isolating and purifying biological materials. The process according to the present invention can be used to isolate nucleic acids or protein from a wide range of organisms across the various kingdoms. The process involving basically 3 steps namely lysis, clean-up and harvest, 3 solutions comprising 6 chemicals and a single changing of sample tube. The process offers a numbers of advantages including fast, high yield, good quality and purity products, flexible in scaling, environmental friendly, low cost and easier to perform