Questions and Answers

The cell theory was proposed by Schleiden and Schwann.

It is defined as the ability of a plant cell to regenerate into a whole plant.

The property of cell totipotency is exploited to culture the plant cells.

The most extensively used nutrient medium is MS medium which was developed by Murashige and Skoog in 1962.

Auxins

Dimethyl sulfoxide (DMSO), glycerol, peoline, and mannitol.

Agrobacterium tumefaciens is the bacteria known as the natural genetic engineer of plants.

The first inter-genetic somatic hybrids between potato and tomato are known as “Pomatoes” or “Topatoes”.

Callus cultures are used for:
(a) Plant regeneration
(b) Preparation of single cell suspensions and protoplasts
(c) Genetic transformations studies

Cell suspension cultures are used for:
(a) Induction of somatic embryos/shoots
(b) In vitro mutagenesis and mutant selection
(c) Genetic transformation
(d) Production of secondary metabolites

Explant Culture: Culture of any plant part such as stem, leaf, cotyledon, etc.
Callus Culture: An unorganized mass of parenchymatous cells used for regeneration, single cell suspension, and transformation.

In vivo gene banks: Preserve genetic resources using seeds, propagules, etc.
In vitro gene banks: Use cell/tissue culture methods to maintain germplasm.

Fusion of somatic cells (protoplasts) from different sources to obtain hybrids. Fusion products are selected using dyes or biochemical markers.

Alkaloids, tannins, resins, latex, etc.
Example: Shikonin from Lithospermum erythrohizon, Quinine from Cinchona officinalis

Agrobacterium transfers T-DNA from its plasmid into plant cells causing crown gall disease. Ti-plasmid is used to insert foreign genes into plant genomes.

Direct gene transfer is inserting genes into cells without vectors. Methods:
(a) Chemical-mediated (PEG)
(b) Microinjection
(c) Electroporation
(d) Particle gun (gene gun)

Plant bioreactors avoid mechanical stirrers as plant cells are shear-sensitive. Oxygen is supplied by bubbling gas gently.

Golden rice is genetically modified rice rich in pro-vitamin A (carotenoids). Normal rice lacks sufficient vitamin A.

The first genes used were cry genes (Bt genes) from the bacterium Bacillus thuringiensis.

A surface antigen CD3 exposed on mature T-lymphocytes is responsible for allograft rejection.

OKT3 (anti-CD3 monoclonal antibody) removes antigen-bearing T-cells from circulation and graft by targeting the CD3 surface antigen.

The most commonly used enzymes are Trypsin and Collagenase.

The first drug produced by mammalian cell culture is Tissue Plasminogen Activator (tPA).

Cell-based therapy uses stem cells to replace damaged or dead cells for treating diseases like blood cancer and neurodegenerative disorders.

• tPA from CHO cells – for heart attack treatment
• Factor VIII – for Haemophilia A
• FSH – for fertility treatment

Animal cells grow in simple glass/plastic containers. Unlike microbial/plant cells, they grow only for limited generations and exhibit contact inhibition.

Chimeric mice are produced by injecting embryonic stem (ES) cells from one mouse (e.g., black) into the embryo of another mouse (e.g., albino). The resulting progeny shows mixed traits like black and white coat color.

Plant cells can grow indefinitely in culture with nutrient replenishment, while animal cells grow for limited generations and show contact inhibition. Animal cells also lack cell-cell/matrix interaction like in vivo.

Embryonic stem cell culture involves maintaining inner cell mass (ICM) cells from mouse embryos in vitro. These cells remain undifferentiated, retain embryo-like characteristics, and can be used to create chimeric mice.

There are two categories:
Finite cell lines: Limited lifespan, grow as monolayers, show contact inhibition.
Continuous cell lines: Transformed, indefinite growth, rapid doubling, grow in monolayer or suspension.

Hybridoma technology fuses antibody-producing B cells with myeloma cells to create immortal hybrid cells that produce monoclonal antibodies. Developed by Köhler and Milstein using PEG.

Karyotyping identifies chromosomal abnormalities and confirms species origin. Normal karyotypes are ideal for transgenics; stability depends on culture conditions and subculturing methods.

Roller bottles: Used for adherent cells; rotate to expose monolayer to medium.
Spinner cultures: Used for suspension cells; use paddles/stirrers to agitate medium.

Microcarrier beads increase surface area for adherent cell growth and are suitable for spinner flasks. They can be made of dextran or glass.

LAF (Laminar Air Flow) protects samples from contamination and operators from hazardous materials.

• Class I: Protects operator
• Class II: Protects operator and culture
• Class III: For highly pathogenic organisms

Primary cell cultures are time-consuming, need fresh tissue or animals, and results may vary. These are overcome by secondary cultures.

(a) Adherent cells: Roller bottles with microcarrier beads
(b) Suspension cultures: Spinner flasks

Established cell lines have known growth characteristics, consistent media requirements, and predictable responses—making them convenient and reliable for research.

Haemophilus influenzae

Structural genomics

Microarray

Contigs

Expressed Sequence Tags

The genome sequencing projects have revealed that the genomes of very different organisms e.g. mouse and man may be quite similar. 12% of the 18,000 worm genes encode proteins whose biological roles could be inferred from their sequence similarity to yeast genes and vice – versa i.e. almost a third of the 6000 yeast genes have functional equivalents in worm genes. It is estimated that the difference between human and chimpanzee genomes is only 1 to 3%. These similarities suggest that neither genome has changed much since we shared a common ancestor 100 million years ago.

Functional genomics- this is a field of study which co-relates the structure and sequence of the genome to its function. In this field the information provided by structure genomics is used to study the function of genes or protein in a systematic manner.
Structural Genomics- In structural genomics, one studies the sequence-structure relationship of a genome. It represents an initial phase of genome analysis which involves creating high resolution genetic physical and transcript maps and ultimately sequencing the genome. Because of rapid advances in the field of proteomics the study of three dimension structure of all proteins has also become easier because of structural genomics.

The genetic variations are caused due to single nucleotide polymorphisms or SNPs which can occur both in the coding and non-coding regions of the genome. It is believed that SNPs occur at 1.6 million to 3.2 million sites in the human genome and may affect gene function, depending upon exact base change and where it occurs.

The usual approach taken by standard computer programs like sequence search programs scan the first 20 symbols. If the symbols encountered switch between any of the 4 base only, then the sequence at hand is taken as a DNA sequence. Instead of T if U is encountered, then it is a RNA sequence. But if the symbols switch between any of the 20 (other than 4), then it is taken as protein sequence.
The usual approach taken by standard computer programs like sequence search programs scan the first 20 symbols. If the symbols encountered switch between any of the 4 base only, then the sequence at hand is taken as a DNA sequence. Instead of T if U is encountered, then it is a RNA sequence. But if the symbols switch between any of the 20 (other than 4),

The Human Genome Project officially started on 1 Oct, 1990 in the United States, as a 15-year program to map and sequence the complete set of human chromosomes and those of general model organisms. It aimed to make a series of maps of each human chromosome at increasingly finer resolution by dividing chromosomes into smaller fragments for cloning and sequencing.

FISH is useful in detecting chromosomal abnormalities such as those in chronic myelogenous leukemia (CML) by using fluorescently labeled probes to identify translocations like the 9-22 Philadelphia chromosome. Red and green fluorescent tags highlight the abnormal chromosomes under a microscope.

Reasons include identifying all genes, understanding gene relationships, providing tools for experiments, organizing genetic data, and archiving the full genetic makeup of the organism for future reference.

Microarray can compare gene expression in normal vs. cancerous cells by converting mRNA to fluorescently labeled cDNA. The labeled cDNAs hybridize to DNA spots on the microarray slide, revealing which genes are upregulated or downregulated in different conditions based on fluorescence color patterns.

Molecular evolution studies changes in DNA and protein sequences over time. Observations include non-random amino acid replacements favoring similar properties, resistance to replacement in some residues like tryptophan, and derivation of PAM matrices for evolutionary comparisons.

Microarray can be used to study tissue-specific gene expression, disease-related regulatory defects, cellular responses to environmental changes, cell cycle variations, and for discovering new drugs.

SNPs (Single Nucleotide Polymorphisms) are variations at a single base pair position in DNA and occur approximately once every 800 base pairs. Despite humans sharing 99.8% of their DNA, SNPs account for the 0.2% variation. These mutations occur in both coding and non-coding regions and are valuable molecular tools for DNA fingerprinting, disease susceptibility studies, and population genetics. For example, a SNP in the ApoE gene is associated with Alzheimer's disease.

A SNP map represents the position of single nucleotide polymorphisms across the human genome. These maps help in locating genetic variations. It is estimated that there are between 1.6 to 3.2 million SNP sites across the human genome.

Alzheimer’s disease is an example of a disease whose susceptibility has been linked to SNPs. A single base change in the ApoE gene is associated with increased risk of developing the disease.

DNA sequences are always read in the 5' to 3' direction.

The C-value paradox refers to the observation that genome size does not correlate with organismal complexity. Higher organisms may not necessarily have more genes than simpler ones, indicating a large amount of non-coding DNA.

Bioinformatics is the management and analysis of biological information stored in databases. It emerged prominently in the late 1980s when researchers began using computers as central repositories for biological sequences, allowing remote access and analysis of data.

Ref Seq is a curated and verified database containing mRNA and protein sequences for human, mouse, and rat. It is widely used for designing gene chips and annotating sequence features of the human genome.

BLAST (Basic Local Alignment Search Tool) is a family of programs used to compare a query sequence against sequences in a database. It uses scoring matrices to reward matches and penalize mismatches, helping identify evolutionary relationships and functional similarities. Top matches can be further explored via tools like Entrez at NCBI.

Directed Sequencing: Uses BAC (Bacterial Artificial Chromosome) vectors to create contigs through overlapping sequences. These contigs are then sequenced in a stepwise manner.
Random Shotgun Sequencing: Involves random fragmentation of the genome into 2–10 kb pieces, cloning into vectors, sequencing, and then assembling based on overlapping sequences.

Database retrieval tools include Entrez, Taxonomy Browser, and Locus Link. Entrez is an integrated system that allows access to literature, sequences, and molecular structures. The Taxonomy Browser helps in exploring the classification of various organisms.

• EMBL: Nucleotide sequences
• SWISS-PROT: Annotated protein sequences
• PDB: 3D structures of proteins
• Ribosomal RNA database: rRNA subunit sequences
• PALI database: Phylogenetic analysis and protein alignments

Based on their origin, DNA sequences in databases are categorized as:

• cDNA: Represents expressed genes; this information is indicated during entry and retrieval.
• Genomic DNA: The most common type of sequence entered.
• ESTs (Expressed Sequence Tags): Partial cDNA sequences; found in the dbEST database; useful for studying gene expression levels.
• GSTs: Sequences from Plasmodium falciparum using mung bean nuclease to cut between genes, sequenced from either end like ESTs.
• Organelle DNA: Includes DNA from mitochondria and chloroplasts, separate from genomic DNA and must be labeled specifically.
• Other Molecules: tRNA and small RNAs are also submitted into databases.

The bioinformatics nomenclature follows recommendations by the International Union of Pure and Applied Chemistry (IUPAC), ensuring global consistency.

• G, A, T, C: Standard nucleotide representations.
• Ambiguity Codes: Used when sequencing results are unclear. For example, if the base could be either G or C but not A or T, the symbol S is used.
• Double-Stranded DNA: Some ambiguity symbols are used because they have the same meaning on both strands.

This system helps accurately represent uncertain base positions in sequences during computational analysis and database entry.

The following are standard IUPAC codes for nucleotide representation:

• a) Guanine: G
• b) Adenine: A
• c) Thymine: T
• d) Cytosine: C
• e) Purine (A or G): R
• f) Pyrimidine (C or T): Y
• g) Amino (A or C): M
• h) Keto (G or T): K
• i) Strong Hydrogen bonds (G or C): S
• j) Weak Hydrogen bonds (A or T): W
• k) Not G (A, T, or C): H
• l) Not A (T, G, or C): B
• m) Not T (A, G, or C): V
• n) Not C (A, G, or T): D
• o) Any base (A, T, G, or C): N

Various types of analyses that can be done using bioinformatics tools include:

• a) Processing raw information - Converting sequence data into genes, proteins, and regulatory elements.
• b) Gene prediction - Tools like GeneMark (for bacterial genomes) and GENSCAN (for eukaryotes) are used.
• c) Protein prediction - Inferring protein sequences from predicted genes using computer programs.
• d) Regulatory sequences - Identifying and analyzing gene regulatory elements.
• e) Phylogenetic analysis - Constructing phylogenetic trees from evolutionary distance and sequence alignment.
• f) Gene discovery - Predicting functions of unknown genes based on existing databases and tools.

Margaret Dayhoff significantly contributed to molecular evolution by:

• Observing that protein sequences evolve in specific patterns, not randomly.
• Noting that amino acids with similar physico-chemical properties often replace each other.
• Identifying that certain amino acids like tryptophan are rarely replaced.
• Developing the PAM (Point Accepted Mutation) matrix based on homologous sequence comparison, laying the groundwork for modern sequence alignment techniques.

The EST (Expressed Sequence Tag) technique was discovered by Craig Venter and his research group.

Intellectual Property (IP) includes patents, trade secrets, copyrights, and trademarks. In biotechnology, IP protects processes and products arising from genetic engineering and recombinant DNA technologies.

Intellectual Property Rights (IPR) refer to the legal rights granted by each country to protect this property, preventing others from making, using, copying, or selling the protected material.

The benefits of obtaining a patent include:

• Encourages and protects intellectual and artistic innovations.
• Accelerates the dissemination of new technologies and ideas.
• Promotes domestic and international investments.
• Ensures inventors benefit from their work.
• Facilitates global distribution of innovation benefits proportionate to national development levels (OECD, 1989).

Breeder’s Rights (PBRs) provide exclusive marketing rights for newly developed plant varieties. Key measures include:

• The FAO’s International Treaty on Plant Genetic Resources, which includes farmers' rights.
• India’s Plant Varietal Protection and Farmer’s Rights Act, 2001 that grants rights to farmers, breeders, and researchers and restricts GURT-based imports like Monsanto’s terminator technology.

To qualify, a new variety must be:

• Commercially new
• Biologically distinct
• Uniform and stable across generations
• Taxonomically valid

A patent is made up of three key parts:

• Grant: A signed, unpublished document that grants legal rights to the inventor.
• Specifications: A published narrative explaining the invention's subject matter and process.
• Claims: Defines the boundaries and protection scope of the invention.

Filing a Patent: The inventor first files in their country, then internationally. A patent attorney helps structure, file, and manage the legal documentation.

• WIPO: World Intellectual Property Organisation
• TRIPS: Trade-Related Intellectual Property Rights
• GM: Genetically Modified
• PCT: Patent Cooperation Treaty
• GURT: Genetic Use Restriction Technology

The European Patent Office (EPO) and other global authorities allow patents on genetically engineered life forms. Examples include:

• Onco mouse – initially rejected but later patented on appeal.
• Genetically engineered E. coli used for producing insulin, growth hormone, and t-PA.
• Transgenic crops like herbicide-resistant cotton and insect-resistant tobacco.

Countries like the USA, Japan, and those in Europe have adapted patent laws to include transgenic organisms.

Trade Secrets: Confidential materials or methods providing business advantage. In biotech, includes hybridization conditions, cell lines, research plans, etc. If leaked prematurely, compensation and legal penalties apply.

Trademarks: Distinctive signs or symbols identifying a company's goods or services. In biotechnology, common for equipment and vectors. International agreements ensure cross-border trademark protection.

Safety concerns in biotech include:

• Proper disposal of microbial biomass and effluents.
• Potential toxicity and allergenicity of microbial products.
• Spread of antibiotic resistance.
• Risk of pathogenicity from genetically modified organisms to humans, animals, and plants.

Patent Co-operation Treaty (PCT): Signed in 1970 and managed by WIPO. It streamlines the international patent application process. PCT handles the “international phase” involving formalities, art searches, and publication before national filings.

Budapest Treaty (1980): Recognizes microorganism deposits in International Depository Authorities (IDAs). Required for patenting biological materials. Deposited strains are catalogued with accession number, isolation source, and function for global use.

Copyright protects the expression of ideas (not the idea itself). In biotechnology:

• It applies to DNA sequence data in published form.
• Protects digital content like databases, software, photomicrographs, and instruction manuals.
• Complements patents and trade secrets by covering expressed formats like printed and digital materials.