Science: Uncovering and Discovering

A zygote inherits a haploid set of chromosomes from both parents. Hence, its phenotype is supposed to result from co-expression of both alleles received from the sperm and the ovum. However, in mammals, gametes do not contribute equal genetic functions to the embryo due to the phenomenon of genomic imprinting. (more…)

As discussed in last blog, genome sequencing has revolutionized several fields in biology, antibiotic target discovery being one of them. Antibiotic targets can be of 2 types:
1) Proteins those are essential for bacterial growth.
2) Proteins those are required for virulence.
Such proteins can be identified by mutating all the genes from a pathogen, one at a time, and observing for the desired phenotype. STM is still another approach (discussed in previous blog). All these approaches, however, are labour intensive. (more…)

Advent of genome sequencing has revolutionized biology to its base. Development in approaches towards vaccine design is a good example of it. Vaccines have come a long way since their discovery by Jenner, a time when even their mode of action was unknown. Major development in vaccination then came up with the advent of recombinant DNA technology, which allowed selection and production of pure immunogens from pathogenic organisms. (more…)

The basic yeast two-hybrid system has been a widely used approach for general protein-protein interaction studies (as discussed in previous blog). With time researchers have modified it in many ways for certain special proteins that are not amenable to the basic system. Some of the modifications can even detect protein-nucleic acid and protein-ligand interactions. (more…)

The basic yeast two-hybrid system is based on the fact that eukaryotic transcriptional activators consist of a DNA binding domain (BD) and an activation domain (AD). Both these domains can be separately fused to 2 different polypeptides X and Y. if X and Y interact with each other, they can bring together BD and AD. This interaction can be monitored by transcriptional activation of a suitable reporter gene. (more…)

Application of proteomic techniques like 2-D gel electrophoresis (2-D GE) directly to crude cell extracts leads to loss of several protein constituents. This is due to the necessity of generalizing conditions for accommodating majority of proteins, rather than using conditions for a specific class of proteins. The resolution also decreases due to the broad range of separation parameters used in such techniques. These shortcomings have led researchers to use prefractionation techniques for enrichment of a particular group of proteins before separation by 2-D GE. (more…)

With increasing availability of whole gnome sequences, a new challenge has arised, that of understanding the function of each gene product, such as role in disease progression. The traditional methods involving testing mutants individually in animal models cannot handle genome-wide information. This has led to development of high throughput signature tagged mutagenesis (STM) approach. (more…)

One of the major challenges in bacterial expression profiling is the requirement for large amount of total RNA. This has led to development of amplification methods that can decrease the amount of starting material required and at the same time maintain the relative proportion of different mRNAs constant. (more…)

In the previous blog extraction of bacterial mRNA for global gene expression analysis was discussed. The next step in the analysis involves synthesis of cDNA from mRNAs before hybridization to microarray reporters. In eukaryotes this is relatively easy as mRNA molecules are polyadenylated, allowing use of oligo(dT) primers. For prokaryotes random hexamers have to be used. However, this may lead to inclusion of undesirable mRNAs, say from tissue/cells of interacting partner in case of interaction studies. (more…)

Success of microarray technology depends largely on extraction of adequate quantities of intact RNA. The problem of RNA concentration compounds especially for the low abundance mRNA species. While studying virulence-associated bacterial genes, this is directly dependent on the number of bacteria recoverable from the infection model. (more…)

One of the most intriguing questions about X-inactivation is how two X chromosomes with similar sequences can have exactly opposite transcriptional state. This property was ultimately linked to a particular region, X-inactivation center (xic), on X chromosome. This linkage was deduced from the outcome of X-to-autosome translocations. Xs that retained xic could still undergo X-inactivation, while those that lose this region could not. (more…)

Heterochromatin consists of the darkly stained regions of chromosome in the interphase nucleus. It is commonly found near the nuclear membrane. Heterochromatic DNA is highly condensed and is frequently associated with centromeres, telomeres and satellite sequences. The genes in such regions are normally in silenced state. (more…)

In the last blog, mechanism of X chromosome silencing in mammals was discussed. It has several implications apart from the dosage compensation described earlier. During early stage of female development, both Xs are active. Since inactivation is random, some cells inactivate paternal X, while others inactivate maternal X. Once a chromosome is inactivated, the inactivation is maintained in all the descending cells. As inactivation is brought about early in development, the adult female is a random patchwork of clonally derived cells, each cone expressing the same X-linked alleles. (more…)

We know that sex of a mammal is determined by its sex chromosome content, XY being male and XX being female. Since Y chromosome carries only a few genes, it is reasonable to assume that females have double the number of X-linked genes as compared to males. Yet females synthesize the same amount of X-linked protein products as male cells. Clearly, some form of dosage compensation mechanism must exist to account for this. (more…)

Although oligonucleotide-bases arrays are gaining popularity, PCR product-based array is a much more developed and established technology. The algorithms for PCR primer design are more advanced than those for oligonucleotide design. (more…)

Microarrays have a limitation of requiring high concentration of target DNA for efficient hybridization. Microelectronic arrays overcome this through the use of electronics. They are based on application of direct current (positive) to individual microelectrodes beneath the test sites. This causes rapid concentration of negatively charged nucleic acids over selected locations. These molecules can then be hybridized to previously addressed and attached nucleic acids at the test sites.
Such electronic addressing allows improvement in both time and efficiency of hybridization process. Reversal of electric potential then allows rapid removal of unhybridized molecules. Thus, the system allows simultaneous analysis of a single sample at multiple test sites, with the provision of washing unbound sample at desired stringency.

Genome atlas is a positional plot of some generally informative parameters and can be used to identify unique regions or specific features in a given genome. It is available in public databases for fully sequenced genomes. Several parameters can be represented on genome atlas: repeats, structural parameters, base composition, gene expression etc. (more…)

Enzymes are immobilized by attaching to or enclosing in inert support materials. Immobilization can be brought about by one of several procedures like physical entrapment, ionic or covalent bonding, and encapsulation. Properties of immobilized enzymes are very different from those in free solution. This depends, to a great extent, on the method of immobilization and the nature of support material. E.g., some harsh immobilization processes can denature the enzyme, leading to reduction in its activity. (more…)

In a previous article, Michaelis-Menten equation for single-substrate reactions was described. However, most biochemical reactions involve at least two substrates, so it is necessary to consider the kinetics of such reactions. A simple example of such reactions is a two-substrate two-product reaction, which can be represented as:

AX + B = BX + A (more…)

Mechanism of enzyme catalysis is very similar to that of ordinary chemical catalysts. Firstly, binding of substrate molecules in close proximity on the enzyme surface increases their effective concentration and hence, the collision rate. The enzyme may also ensure that the reacting groups of the bound substrates are in correct orientation to each other. Next Enzyme also stabilizes transition state, thus reducing the energy of activation. (more…)