eChapters

Biological sequences like the nucleotide and amino acid sequences can be analyzed by means of the sequence alignment. The objective behind this is to obtain knowledge regarding the similarity between the query and the target sequence to decipher the structure, function, homology of the query sequence.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

Two types of molecular weight (MW) standards are routinely used. The Lambda/HindIII and PhiX174/HaeIII MW standards provide a useful reference for calculating molecular weights of large and small DNA fragments, respectively, after electrophoretic separation; the “internal MW standards” provide a means for normalizing fragment migration distances within each lane to facilitate comparisons between lanes on the same or different luminographs in fingerprinting studies.

ANN is a Mathematical model or Computational model based on biological neural networks. It consists of an interconnected group of artificial neurons and processes information using a connectionist approach to computation. Neural Networks are non-linear statistical data modeling tools. That can be used to model complex relationships between inputs and outputs or to find patterns in data.

Insights into the three-dimensional (3D) structure of a protein are of great assistance when we plan experiments to understand protein function and in assistance of the drug design process.

Homology modeling
    
·    Homology modeling consists of the extrapolation of the structure for a new (target) sequence from the known 3D-structure (X-ray crystallographic or NMR) of related family members (templates).
    
·    Homology modeling is for those targets that have homologous proteins with known structure (>30% identity)
    
·    Homology modeling is for easy targets.
    
·    Homology modeling treats the template in an alignment as a sequence and only sequence homology is used for prediction.
    
·    When the sequence identity between the target and template sequence is low (<25%), homology modeling may not produce a significance result.

In recent years, we have witnessed an explosion of biological information and the bioinformatics data arising from the rapid research and unprecedented progress in molecular biology. The novel genomic technologies have revolutionized the drug designing, discovery and development processes in government as well as private pharmaceutical industries through the use of these technologies. Various databases are doubling in size every year and fortunately we have the complete genome sequences of more than 100 organisms. At present, the pharmaceutical industries have clasped genomics as a source of drug targets. They also distinguish that the field of bioinformatics is crucial for validating these potential drug targets and for determining the ones that are most suitable for entering the drug development pipeline. Due to our increased understanding of molecular biology, there has been a change in the way that medicines are being developed. Once upon a time, new synthetic organic molecules were tested in animals or in whole organ preparations. Now this approach has been replaced with a molecular target in which in-vitro screening of compounds against purified, recombinant proteins or genetically modified cell lines is carried out with a high throughput as a consequence of better and ever improving knowledge of the molecular basis of disease.

ABSTRACT

This paper is an attempt to draw the attention of readers of this volume to understand the importance of documentation and preservation of earth’s resources.  

Introduction

Earth has innumerable treasures that were and are being exploited by mankind. Geo-resources in the form of minerals, non-renewable hydrocarbons, paleontologic relics and related evidences indicate earth’s evolutionary history. Continuum of biological activity albeit varying rates of proliferation had resulted in accumulation of natural resources embedded in Earth and distributed at specific geographical niches. Biological perspective of earth history is significant in unraveling bio-geo-sphere interactions through geological time. Fossil records of invertebrates, vertebrates and plants are signals of major changes in earth’s evolutionary history. These constitute unparalleled windows to understand continental separation. Intercontinental and intra continental migration of living forms are also traced through fossil study.

This statement signifies that the casualty of people is not due to the earthquake but due to the damage of buildings by earthquake forces. In developed countries, the casualty and loss of property is very minimal in comparison to the death toll and losses in India in the event of earthquakes of same magnitude. It has been experienced in the past that on the event of earthquakes of Richter magnitude scale of 6 or more, the death toll in India is in terms of thousands where as in Western Countries, it is multiple of tens. The less casualty is due to less damage of buildings and other infrastructures which in turn is due to the design and construction of Buildings and other structures as per the earthquake resistant theory. This paper discusses the issues related to the earthquake hazards in India and the concept of earthquake resistant structures.

Macromutations were observed in the present set of experiments, under all the treatments (Table 12-14).

As the dosage of gamma rays increased, there was a decrease in their frequency. EB showed less mutation frequency than gamma rays and EMS. But in combination with gamma rays it produced more frequency of macromutations. As the dose increased the frequency also increased. EMS as single treatment produced macromutations like that of gamma rays, but in combination with gamma rays it produced more frequency than gamma rays as individual treatment. However, as the dosage increased the mutation frequency was also reduced. Medium dose combination produced more mutations than higher and lower dose combinations of gamma rays + EMS.

On M₂ plant basis, gamma rays in combination with EB produced the maximum viable mutation rate (4.04), whereas gamma rays individually induced a maximum rate of 2.23 and EB as a sole mutagen generated a maximum rate of 0.79. But EMS as a single treatment showed 1.69 as viable mutation rate, in combination with gamma rays it generated as much as 2.49. Combination treatment showed a less than synergistic effect.

Introduction

Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixtures. The method is based on the separation of proteins according to their size and then locating by binding them to a dye. It is particularly useful for monitoring protein purity and determines their relative mass. The SDS-PAGE enables the students to understand the theory behind the techniques in checking the purity of the protein and identifying the protein in a test sample.

Separation of Proteins

Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Minisatellite is a section of DNA that consists of a short series of bases 10– 60bp. These occur at more than 1000 locations in the human genome. Some minisatellites contain a central (or “core”) sequence of letters “GGGCAGGANG” (where N can be any base) or more generally a strand bias with purines (Adenosine (A) and Guanine (G)) on one strand and pyrimidines (Cytosine (C) and Thymine (T)) on the other. It has been proposed that this sequence per se encourages chromosomes to swap DNA. In alternative models, it is the presence of a neighbouring cis-acting meiotic double-strand break hotspot which is the primary cause of minisatellite repeat copy number variations. Somatic changes are suggested to result from replication difficulties (which might include replication slippage, among other phenomena). When such events occur, mistakes are made, this causes minisatellites at over 1000 locations in a persons genome to have slightly different numbers of repeats, thereby making them unique. The most highly mutable minisatellite locus described so far is CEB1 (D2S90) described by Vergnaud.

Since the mid 1980s, genome identification and selection has progressed rapidly with the help of PCR technology. A large number of marker protocols that are rapid and require only small quantities of DNA have been developed. Three widely-used PCR-based markers are RAPDs (Williams et al., 1990), SSRs or microsatellites (Tautz, 1989), and AFLPs (Vos et al., 1995). Each marker technique has its own advantages and disadvantages. RAPD markers are very quick and easy to develop (because of the arbitrary sequence of the primers) but lack reproducibility (Karp et al., 1997; Hansen et al., 1998; Jones et al., 1999; Virk et al., 2000). AFLP has medium reproducibility but is labour intensive and has high operational and development costs (Karp et al., 1997). Microsatellites are specific and highly polymorphous (Karp et al., 1997, Jones et al., 1999), but they require knowledge of the genomic sequence to design specific primers and, thus, are limited primarily to economically important species.

The choice of a molecular marker technique depends on its reproducibility and simplicity. The best markers for genome mapping, marker assisted selection, phylogenic studies, and crop conservation have low cost and labour requirements and high reliability. Since 1994, a new molecular marker technique called inter simple sequence repeat (ISSR) has been available (Zietkiewicz et al., 1994). ISSRs are semiarbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in the presence of nonanchored primers also has been called microsatellite-primed PCR, or MP-PCR, (Meyer et al., 1993).

Knowledge on genetic variability of the available population is very essential for any crop improvement programme, as it will positively enhance the efficiency of selection. The variability in quantitative characters increase considerably by treating the biological materials with different mutagenic agents. It is possible to identify mutants for different characteristics which can be useful in further programme. Variability is measured by different statistics viz., phenotypic coefficient of variation (PCV) and genotypic co-efficient of variation (GCV). GCV would be more useful for the assessment of inherent variability as it exhibits the heritable portion only.

1. Introduction:- Overview of Good Laboratory Practices (GLP)
1.1 Definition and Scope of GLP
GLP refers to a set of standards that govern the organizational processes, personnel responsibilities, and conditions under which laboratory studies are conducted. It aims to regulate research environments and ensure the generation of high-quality, traceable, and reproducible results. The scope of GLP encompasses a wide range of scientific fields including pharmaceuticals, agrochemicals, veterinary products, and environmental safety assessments. Good Laboratory Practices (GLP) regulations were developed by the Organization for Economic Co-operation and Development (OECD) and well adopted by regulatory agencies world-wide. The primary objective of GLP is to promote high standards of accuracy and integrity in non-clinical research to protect human health and the environment.
1.2 Principles of GLP
The fundamental principles of GLP include:
• Study Planning and Protocol Development: Every study must have a clear plan and defined objectives, which should be outlined in a comprehensive protocol.
• Personnel Responsibilities: The roles of study directors, scientists, quality assurance personnel, and support staff are clearly defined.
• Test Systems and Facilities: Laboratories must maintain appropriate facilities and control systems for conducting studies. These include properly calibrated equipment, controlled environments, and standardized procedures.
• Data Integrity and Reporting: Data must be meticulously recorded, verified, and archived, ensuring it is traceable and reproducible.
• Study Documentation and Archiving: All records, raw data, and reports must be stored and accessible for a period defined by regulations, ensuring transparency and reproducibility.

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

Mitochondria are key energy generators in most eukaryotic cells. In recent years a number of mitochondrial genomes have been completely sequenced, contributing to the knowledge of molecular features related to function and evolution of this peculiar genome (Boore, 1999). The pathways of mitochondrial DNA evolution are obscured by their extraordinary size. Gene content, gene rearrangement and mode of expression are the characteristics of mitochondrial genomes. The mitochondrial genomes from eukaryotes reveal large size differences between animal, fungal and plant species. The average size of animal mtDNA is ~ 16 kb (Boore, 1999), whereas plant mtDNAs range from 200 to 2500 kb (Palmer, 1990). The size of fungal mtDNA’s range from 19 kb to 176 kb (Hudspeth, 1995).The mtDNA genome has a limited number of protein coding genes such as NADH ubiquinone oxidoreductase (ND2 to ND5), cytochrome c oxidase (COX), succinate dehydrogenease (SDH), ATP synthase (ATP6), cytochrome (CYTB), etc. Among non-coding genes only rRNA and tRNA genes are found.Ribosomal RNA (rRNA) is a component of the ribosomes, the protein synthetic factories in the cell. rRNA molecules are extremely abundant. They make up at least 80% of the RNA molecules found in a typical eukaryotic cell. Synthesis of the three nucleolar rRNA molecules is unusual as they are made on one primary transcript that is chopped up into three mature rRNA molecules.

Introduction

The most common method of bacterial transformation utilizes high levels of calcium chloride to facilitate the entry of plasmid DNA vectors into E.coli in conjunction with structural alteration of the bacterial cell wall. In general, there are three basic steps in the introduction of plasmid DNA unto cells.

1. Preparation of “competent” cells to enable cells to accept DNA.
2. Transformation of these competent cells, and
3. Selection of transformants.

Most E.coli strains exhibit transformation efficiency (measured as transformants per microgram plasmid DNA) in the range of 10⁵ to 10⁸.

The factors influencing this efficiency depends upon the condition that render cells competent.

In the context of molecular biology, a consensus sequence is the minimum nucleotide sequence that shows the characteristics common to most members of the family among a set of related genetic sequences.It serves in representing the results of a multiple sequence alignment, where related sequences are compared to each other, and similar functional sequence motifs are found. It also shows conserved/variable residues. It is found several times in the genome and is thought to play the same role in its different locations. For example, many transcription factors recognize particular consensus sequences in the promoters of the genes they regulate. In the same way restriction enzymes usually have palindromic consensus sequences, usually corresponding to the site where they cut the DNA. Transposons act in much the same manner in their identification of target sequences for transposition. Splice sites, i.e. those sequences immediately surrounding the exon-intron boundaries, can also be considered as consensus sequences. Defining a consensus DNA binding site by examining consensus sequences is a key work carried out by biologists (Harley et al., 1994). Rareness or anomaly detection followed by in depth analyses in such sequences may reveal significant information regarding the structural, functional and biochemical mechanisms of the genes which are of much importance to biologists.

Introduction

The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg^- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme.

Mutagenic Agent

Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10^-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

Neisseria meningitidis, also simply known as meningococcus, is a gram-negative bacterium best known for its role in meningitis. It only infects humans; there is no animal reservoir. It is the only form of bacterial meningitis known to cause epidemics. The carbohydrate capsule of N. meningitidis determines its virulence and is targeted by the immune system. Approximately 12 strains of N. meningitidis exist and are characterized by the polysaccharide expressed on its capsule: A, B, C, 29-E, H, I, K, L, W-135, X, Y and Z. Inhibition of Pencillin Binding proteins (PBPs) leads to irregularities in cell wall structure such as elongation, lesions, loss of selective permeability and eventual cell death and lysis. 3-D structure of PBP was studied using Bioinformatics tool. Using Homology modelling, converted protein sequence of penicillin binding protein, extracted from NCBI server, in FASTA format and performed PDB-BLAST to search a desired template. Selection of template was based on lower E-value, bit score and identity. Using MSA with the help of CLUSTAL W software conserved region was found between the target and template sequence, 1K25. The 3-D structure of penicillin binding protein was found with the help of Modeller 9v2 and molecular dynamics was performed using GROMACS software. The docking was performed (Autodock 3.0 software) using 3 ligands viz. Cefuroxime, Chloramphenicol, Cefalotoxime with standard Bioinformatics tools.