- Who proposed the concept of the “cell theory”?
The cell theory was proposed by Schleiden and Schwann.
- Define Cell-totipotency.
It is defined as the ability of a plant cell to regenerate into whole plant.
- What is the basic technique in plant tissue culture?
The property of cell totipotency is exploited to culture the plant cells.
- Which is the most commonly used culture medium for plant cells and tissues?
The most extensively used nutrient medium is MS medium which was developed by Murashige and Skoog in 1962.
- Name the substance added to the culture medium to induce callus induction.
- Name a few compounds used as cryoprotectants.
Dimethyl sulfoxide (DMSO), glycerol, peoline and mannitol.
- Name the bacteria known as natural genetic engineer of plants
Agrobacterium tumefaciens is the bacteria known as natural genetic engineer of plants.
- What is the name of the first inter-genetic somatic hybrids between potato and tomato?
The first inter-genetic somatic hybrids between potato and tomatoes are known as “Pomatoes” or “Topatoes”.
- Fill in the blanks
(a) --------------- is the gas responsible for the ripening of fruits.
(b) Golden rice is rich in --------------------
(c) ------------------refers to an unorganized mass of cells, which are generally parenchymatous in nature.
(d) Breeding associated with molecular markers (nucleic acid) is known as ---------------------
(e) Genetically engineered ------------- plants produced polyhydroxy butyrate globules in their chloroplasts.
(f) ------------------ is the enzyme which inhibits pollen formation and hence prevents unnecessary pollination.
Ans. (a) Ethylene, (b) Pro-vitamin A, (c) Callus, (d) Molecular Breeding, (e) Arabidopisis, (f) Barnase
- What are the uses of callus culture?
Callus cultures are used for:
(a) Plant regeneration
(b) Preparation of single cell suspensions and protoplasts
(c) Genetic transformations studies
- Write some uses of cell suspension cultures?
The 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
(a) Explant culture, (b) Callus culture.
Explant Culture- The culture of the plant parts (explants) is known as explant culture. The explants can be any part of the plant like the piece of stem, leaf, cotyledon, hypocotyls etc.
Callus culture- Callus refers to an unorganized mass of cells, which are generally parenchymatous in nature. Callus cultures are used for plant regeneration, preparation of single cell suspensions and protoplasts, and genetic transformations.
- What do you mean by in vivo gene banks and in vitro gene banks?
In vivo gene banks have been made to preserve the genetic resources by conventional methods e.g. seeds, vegetative propagules etc. In vitro gene banks have been made to preserve the genetic resources by non conventional methods is cell and tissue culture methods. This ensures the availability of valuable germplasm to breeder to develop new and improved varieties.
- What is the technique of parasexual hybridization?
The parasexual hybridization or somatic cell hybridization offers an alternative for obtaining distant hybrids with useful agronomic traits (known as somatic hybrids or parasexual hybrids) which would never be formed in nature through sexual fertilization. The protoplast is isolated from the two sources and is allowed to randomly fuse with each other. The required fusion products are selected either through visual selection, biochemical methods, and fluorescent dyes.
- What are the natural secondary metabolites produced from plants?
Plants produce thousands of sophisticated chemical molecules. These include the chemicals that are required for the plant’s basic metabolic processes such as alkaloids, resins, tannins, latex etc. Shikonin is obtained from Lithospermum erythrohizon is used as antimicrobial, red pigment used in lipsticks and dye for silk. Quinine is obtained from Cinchonal officinalis is used as antimalarial drug.
- “The Agrobacterium is considered as Natural Genetic engineer of plants.” Comment.
This bacterium is known as natural genetic engineer of plants since these bacteria have the natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti Plasmids can be used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti – plasmid in place of unwanted sequences.
- What is direct gene transfer? What are the methods of direct gene transfer?
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The following are some of the common methods of direct gene transfer in plants:
(a) Chemical mediated Gene transfer- Certain chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.
(b) Microinjection- Here the DNA is directly injected into plant protoplasts or cells using fine tipped glass needle or micropipette.
(c) Electroporation-In this case, a pulse of high voltage is applied for protoplasts/cells/tissues, which makes transient pores in the plasma membrane which facilitates the uptake of foreign DNA.
(d) Particle Gun- In this method, the foreign DNA is coated (precipitated) on to the surface of minute gold or tungsten particles (1-3 micrometer) and bombarded (shot) on to the target tissue or cells using a particle gun (also called as gene gun/shot gun/micro projectile gun).
- What is the specialty of the plant bioreactor?
Plant cells are cultured in specially designed “plant bioreactors” which essentially do not have a stirrer because plant cells are shear sensitive. In place of stirrer, gas is gently bubbled which provides stirring as well as meet the demand of a higher oxygen supply.
- What is Golden Rice? In what way it is different from the normal rice?
Normal rice is very low in vitamin A. Prof. Ingo Potrykus and Dr. Peter Beyer developed genetically engineered rice known as Golden rice which is enriched in pro-vitamin A by introducing three genes involved in the biosynthetic pathway for carotenoid, the precursor for vitamin A. The seeds of Golden rice are yellow in color because of pro-vitamin A which is produced in the entire grain.
- What is the name of the first genes available for genetic engineering of crop plants for pest resistance?
The first genes available for genetic engineering of crop plants for pest resistance were ‘cry genes’ (popularly known as Bt genes) from a bacterium Bacillus thuringensis.
- Which antigen is present on T-cells which cause graft rejection during transplantation?
A surface antigen CD3 exposed on mature T-lymphocytes is responsible for allograft rejection.
- How does OKT3 help in transplantation?
An antibody directed against CD3 surface antigen of T –cells, OKT3 (anti-CD3 Moab) removes antigen bearing cells from circulation and from the graft.
- Which are the most commonly used enzymes for separating cells in a given tissue for culture?
Trypsin and collagenase
- Which is the first drug to be produced by mammalian cell culture?
Tissue plasminogen Activator
- What do you mean by cell based therapy?
The stem cell technology is used to replace damaged and dead cells to treat blood cancer, neuro and degenerative diseases etc. This is known as cell based therapy.
- Name a few medically important commercial proteins produced by animal cell culture.
t-PA protein produced by CHO cells is used to treat heart attack. Factor VIII from CHO cells is used to treat Haemophilia A. FSH From CHO cells is used to treat fertility.
- What is the most distinct feature of animal cell culture?
Animal cells can grow in simple glass and plastic containers. While growing microbial cells and plant cells, if the nutritive medium is changed and the cells are replenished with the culture medium, the cells will grow indefinitely. However, animal cells, depending on the tissue they have been isolated from can grow even in the best nutritive media to only limited generations. The animal cells in culture also exhibit contact inhibition.
- Fill in the blanks:
(a) ------------ was the first drug to be produced by mammalian cell culture.
(b) ------------- are used for scaling up the production of suspension cells.
(c) In----------- the cells adhere to the total curved surface of the microcarrier beads.
(d) An------------- microscope is used for visualizing cell cultures in situ.
(e) ------------------involves the storing of cells at very low temperature.
(f) ---------------------is a highly effective therapeutic agent who reverses acute renal allograft rejection.
(g) ------------------play a major role in rejecting foreign tissue in transplantation.
(h) The animal cells in culture exhibit the phenomenon of ---------------
(i) The maintenance of growth of the cells in culture medium is known as -------------------
(j) ------------------ are those cell lines which have a limited life span and they grow through a limited number of cell generations.
(k) Cell lines transformed under in vitro culture conditions give rise to-------------------
Ans (a) Tissue plasminogen activator, (b)Spinner cultures, (c) Roller bottles, (d) Inverted microscope, (e) Cryopreservation, (f) OKT3, (g) T cells, (h) Contact inhibiton, (i) primary cell culture, (j) Finite cell lines, (k) Continuous cell line
- What is a chimeric mouse?
Embryonic stem cells are cell lines directly derived from the inner cell mass of fertilized mouse embryo with out the use of immortalizing or transforming agents. It fertilized mouse egg is grown in culture (in vitro fertilization), it is observed that after several steps of cleavage, the cells which are dividing get accumulated in one corner. These cells are called inner cell mass (ICM cells) cells. These cells could be maintained in tissue culture in the presence of irradiated fibroblast cells. It was observed that these cells could be used to create chimeric mice. In chimeric mice, it was possible to take ES cells from a black mouse and implant it into the embryo of an albino (white) mouse. The progeny so developed had skin colour of black and white (a chimera).
- What are the differences between plant cell culture and animal cell culture?
The plant cells in culture can grow indefinitely if the nutritive media is constantly replenished. However, the animal cells, depending on the tissues they have been isolated from can grow even in the best nutritive media to limited generations. The animal cells also exhibit contact inhibition i.e. when cells grow and reach the walls of the container they are prevented from further growth. Another important feature of cell growth in culture is that their environment is different from that in vivo. The other major differences include the absence of cell-cell interaction, cell-matrix interaction etc.
- What is Embryonic stem cell culture?
Embryonic stem cells are cell lines derived directly from the inner cell mass of fertilized mouse embryo without use of immortalizing or transforming agents. If we fertilized a mouse egg and grow it in tissue culture, it is observed that after several steps of cleavage, the cells which are dividing get accumulated in one corner. These cells are called inner cell mass (ICM) cells. These cells could be maintained in tissue culture in the presence of irradiated fibroblast cells. These cells could retain the characteristics of the embryo founder cells, even after prolonged culture and extensive manipulation. The cells reintegrate fully into embryogenesis when returned to the early embryo. The cells would be used to create chimeric mice and to maintain a stable euploid karyotype. The cells would renew without differentiating in culture.
- How many categories of cell lines are available?
The various types of cell lines are categorized into two types i.e. finite cell lines and continuous cell lines.
(a) Finite cell lines- Finite cell lines are those cell lines which have a limited life span and they grow through a limited number of cell generations. Finite cell lines show the property of contact inhibition, density limitation and anchorage dependence. The mode of growth is in the monolayer form. The growth rate is slow and doubling time is around 24 to 96 hrs.
(b) Continuous Cell lines- Cell lines transformed under in vitro culture conditions give rise to continuous cell lines. The various properties associated with continuous cell lines are, the ploidy- either aneuploid or heteroploid. There is no contact inhibition and no anchorage dependence. The mode of growth is either monolayer or suspension form. The growth rate is rapid and doubling time is 12 to 24 hours.
- What do you understand by the term hybridoma technology?
Antibodies obtained from serum are a heterogeneous population of proteins released by different populations of B lymphocytes and therefore are known as polyclonal antibodies and bind very specifically to an epitope or an antigen. They are produced from antigen activated B lymphocytes cells that have been immortalized by hybridizing (fusing) them with a myeloma cell (cancerous lymphocyte). Ceaser, Milstein and George Kohler developed the immortalized hybridoma cell line by fusion of antibody producing B cells and cancerous cell (myleoma) in presence of polyethylene glycol (PEG). Thus the hybrid retains the ability of the B cells to secrete antibody and the ability of the myeloma cell to grow indefinitely. The Hybridoma technology has created a revolution in the area of diagnostics and antibody based therapy.
- What is the importance of karyotyping in established cell lines?
Once a cell has been established, its karyotype is determined. This will confirm the species of origin, at least for those cells karyotyped, and determine the extent of gross chromosomal changes in the line. Karyotypes may vary from being near normal tobeing aneuploid. While a normal karyotype is desirable, the presence of an abnormal karyotype is desirable, the presence of an abnormal karyotype does not preclude using the cells for in vitro studies, especially if it has been demonstrated that the cells retain the normal function expected of them.
If a normal karyotype is required e.g. to create transgenic animals, then special care is taken in handling the cells to minimize chromosomal changes. The stability of the karyotype depends on the species from which they are derived, the growth conditions used, the way in which the cells are subcultured, and whether or not the cells are frozen.
- Differentiate between roller bottle and spinner cultures?
In roller bottles the cells adhere to the total curved surface area of the microcarrier beads, thereby markedly increasing the available space for growth. These tissue culture bottles can be used in specialized CO2 incubators with attachments that rotate the bottles along the long axis. After each complete rotation of the bottle, the entire cell monolayer has transiently been exposed to the medium. The volume of the medium need only be sufficient to provide a shallow covering over the monolayer.
Spinner Cultures- Spinner cultures are used for scaling up the production of suspension cells. They consist of a flat surface glass flask with a suspended central Teflon paddle that turns and agitates the medium when placed on a magnetic stirrer. Commercial versions incorporate one or more side arms for sampling and/ or decantation. The cells are not allowed to settle to the bottom of the flask and thus cell crowding occurs only at very high densities.
- What is the use of microcarrier beads?
The microcarrier beads are used to increase the number of adherent cells per flask and are either dextran or glass based . they come in a range of densities and sizes. The beads are buoyant and therefore can be used with spinner culture flasks. The surface area available for cell growth on these beads in huge.
- What functions are performed by LAF? How many types of LAFs are present?
A LAF hood (Laminar Air flow hood) perform two functions-
(i) to protect the tissue culture from contamination by protecting it from the operator (i.e. Sterile environment)
(ii) to protect the operator from the tissue culture (in situation of potential infection risk)
Depending on the nature of the cells and organisms being handled three types of culture hoods are available:
Class I hoods give good protection to the operators and to a lesser degree, the cell culture.
Class II hoods offer protection to both the operator and the cell culture.
Class III hoods are used for work with highly pathogenic organisms.
- What are the drawbacks of using primary cell cultures?
The drawbacks of primary cell culture are that they are time consuming and require use of line animals or fresh tissue. Also there can be considerable variation from one preparation to another particularly if prepared by different people. These difficulties can be overcome by using secondary cell cultures.
- Name the scale up methods used for (a) adherent cell cultures (b) suspension cultures.
The scale up methods includes roller bottles with micro carrier beads for adherent cell cultures and spinner flasks for suspension cultures.
- What are the advantages of using established cell lines for research by the scientists?
The advantages of using established cell lines is that their growth characteristics, media requirements and responses to selected reagents are established and therefore convenient for the scientist to use.
Questions on Genomics
Which microorganism’s genome was first sequenced?
- What is the field of genomic dealing with the sequence structure of DNA called?
- What are cDNAs spots on a glass slide used for expression studies called?
- What are linear DNA sequences or clones having overlapping sequences called?
- What do EST stands for?
Expressed Sequence Tags
- Based on genomic studies, why do people say that different species and organisms had a common ancestor 100 million years ago?
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.
- Differentiate between functional and structural genomics?
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.
- What causes genetic variations in individuals?
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.
- If you are given a sequence with out any label, how will you find out whether it is a DNA sequence or RNA sequence or a 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), 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),
- What is Human Genome Project?
The Human Genome project officially started on 1 Oct, 1990 in the United States, as a 15 year programme to map and sequence the complete set of human chromosomes, as well as those of general model organisms. The strategy of this international project was to make a series of maps of each human chromosome at increasingly finer resolution. In this approach chromosomes were divided into smaller fragments that could be cloned and fragments wee arranged to correspond to their locations on a chromosome. After mapping, each of these ordered fragments would be sequenced.
- How can FISH technique help in the detection of chromosomal abnormalities?
The application of FISH can be illustrated by taking an example of chronic mylegenous leukaemia (CML). It was observed from the karyotype analysis of the lymphocyte preparation made from blood samples of CML patients that there was a 9-22 translocation in the chromosome (Philadelphia chromosome). Although by counting the number of such cells it was possible to find out the severity of the disease, it was not an easy procedure. The regions on the chromosomes involved in translocation were identified on chromosomes 9 and 22 from the DNA library it was possible to pick up clones carrying the particular genes involved in CML. Using nick translation it was possible to fluorescently label chromosome 9 region with red colour and chromosome 22 region with green colour and prepare the probe. It was observed that when CML lymphocytes smeared cells were hybridized with the two probes in situ and when observed under fluorescent microscope, the cells, which were affected, appeared as red and green.
- What are the reasons for completely sequencing a genome?
The reasons for completely sequencing a genome are:
a) it provides a base for the discovery of all inventory of genes.
b) The sequence shows the relationship between genes.
c) It provides a set of tools for future experimentation.
d) The sequencing provides an index to draw and organize all genetic information about the organism.
e) Ultimately the whole genome sequence becomes an archive for further containing all the genetic information required to make the organism.
- How can microarray be used for comparative studies?
If one were to study the effect of certain drugs or say a cancerous cell so as to see which genes are activated or switched off vis-à-vis a normal cell one can use the Microarray technology as a comparative tool. Now in order to study the expression of different genes under different conditions one needs to look into the different mRNA population in the cell since it is the mRNA which gets converted into proteins. mRNA accounts for only 3% of the total RNA of a cell. The m-RNA is first converted into its cDNA by RT PCR since the mRNA is very unstable and prone to attack by RNase in vitro conditions. The cDNA is more stable. Reporter fluorescent molecules are tagged on to the cDNA. Two samples are taken one of a normal person and another of the cancerous person which is to be compared. The normal persons and the cancerous person’s cDNAs are labeled with different (for example Red and Green) fluorescent dyes. The samples are then spotted on to the various DNA spots on the microarray. If there is a match i.e. the sample DNA hybridize with the DNA on the microarray then the spots whose mRNA is present at a higher level in one or the other cell population shows up as predominantly red or green. Yellow spots have roughly equal amounts of bound cDNA from each cell population. This way the whole genome can be monitored for expression studies on a single glass slide.
- What is molecular evolution? What are the observations regarding protein sequence variations vis-a vis evolution?
Zucker Kandle and Pauling (1962) proposed a new approach of studying evolutionary relations using sequence variability. This initiated a new field called molecular evolution. The approach was based on the observation that functionality related homologous protein sequence were similar. It was observed that protein sequences undergo variation during evolution according to certain patterns such as:
a) amino acids were not replaced at random but were altered with specific preferences e.g. amino acids of similar physicochemical characteristics were preferred one over another.
b) Some amino acids such as tryptophan, was generally not replaced by any other.
c) Based on several homologous sequences, a point accepted mutation (PAM) matrix could be developed.
- What are the different areas which can be studied using microarray technologies?
This technique can be used to study the following areas:
1) Tissue specific genomes.
2) Regulatory defects in a disease.
3) Cellular responses to environment.
4) Cell cycle variations.
5) Discovering drugs.
- Write a short note on SNPs and their relevance as molecular tools?
All human beings or Homo Sapiens share 99.8% of their genomic sequence i.e. only 0.2% of the sequence varies between different individuals. One of the most important variations, particularly, because of its use as a molecular tool are point mutation called as SNPs or single nucleotide polymorphisms which occur both in the coding and non coding regions of the genome. They occur in approximately, 1 in every 800 base pairs frequency. They occur when one base at a particular portion differs for different individual. These SNPs are proving to be excellent tools for DNA fingerprinting detecting susceptibility and predisposition to various diseases. Example: a single base difference in the ApoE gene is known to be associated with Alzheimer’s disease.
- What is meant by SNP map? How many SNPs occur in the whole genome?
The position of SNPs on human DNA is depicted by SNP map. Around 1.6 to 3.2 million sites.
- Give example of one disease, whose susceptibility has been located with the help of SNPs.
- In which direction is the DNA sequences always read?
- What does the C-value paradox indicates?
The C-value paradox indicates that despite the increasing complexity in higher animals, the number of genes does not increase likewise.
Questions on Bioinformatics
Questions on Biotechnology and society
- What is bioinformatics?
Bioinformatics is the management and analysis of biological information stored in data bases especially in late 1980’s when researchers started to use computers as central sequence repository, from where the data could be accessed remotely.
- What is Ref Seq?
Ref Seq is a well verified database of mRNAs and proteins of human, mouse and rat. The data provided in Ref Seq has been used in many cases such as designing gene chips and describing the sequence features of the human genome.
- What is the BLAST family of search tools?
The “BLAST” acronym stands for Basic Local Alignment Search Tool. A given sequence in the database using matrices that specify scores to either “reward” a match or “penalize” a mismatch. Top scoring matches are ranked to distinguish between a similarity due to ancestral relationship or due to random chance. True matches are further examined thoroughly with other details accessible through Entrez and other tools available at NCBI.
- What are the differences between directed and random sequencing?
Directed Sequencing of BAC Contigs- BAC vectors are capable of carrying inserts as large as 80-100 Kb. The vectors are used to make Genomic Libraries. The overlapping regions are used to assemble the various insets into continuous contigs. The individual contigs are then sequenced by breaking the DNA and cloning into smaller vectors. Since this is a stepwise directed method it is also called as Directed sequencing.
Random Shotgun Sequencing- In this method the genome is randomly broken up into sizes of 2-10 kb range and inserted into a vector (plasmids). These are then sequenced and ordered with respect to each other with the help of overlapping or common sequences.
- What are database retrieval tools and what are its uses?Among the
database retrieval tools are ENTREZ, taxonomy, BROWSER, LoCUS LINK. Entrez is an integrated database retrieval system. Through this system one can access literature (in the form of abstracts), sequences, and structures. Entrez is an excellent system for obtaining comprehensive information on a given biological question. The taxonomy browser provides information on taxonomic classification of various species.
- Mention all the databases and the type of information available on them?
Database Information available
EMBL (European Molecular Biology Laboratory) Nucleotide sequence
SWISS-PROT Annotated protein sequence
PDB (Protein Database) Three-dimensional structure of proteins
Ribosomal RNA database rRNA subunit sequences
PALI database Phylogenetic analysis and alignment of proteins
- What are the different types of sequences entered into the databases?
Based on their origin the various DNA sequence entries into the databases can be classified into the following types:
cDNA: Many sequences entered are originally cDNA sequences. While entering such sequences one has to mention this fact. Also this information is available when the sequence is retrieved. Thus these are expressed as part of the total sequences.
Genomic DNA: Most sequences entered are Genomic DNA sequences.
ESTs: Stands for Expressed Sequence Tags. These comprises of partial cDNA clones i.e. cDNAs sequence from one end. It has its own special database called the db EST. EST data can give information regarding the expression patterns by counting the number of ESTs corresponding to each gene divided by the total number of ESTs.
GST: In Plasmodium falciparum the enzyme Mung Bean nuclease cuts between the genes. The digested DNA can be used to prepare genomic library. The sequence can be read from either ends as in ESTs.
Organelle DNA: Eukaryotic cell possess cell organelles such as mitochondria, chloroplast. Since they possess their own DNA which is different from the Genomic DNA one has to mention it specifically while entering such data.
Other molecules such as tRNA and smaller RNAs are also entered into databases.
- Write short note on the nomenclature system used in Bioinformatics work?
The nomenclature system that researchers use in bioinformatics work is based on the International Union of Pure and Applied Chemistry recommendations. This ensures uniformity of use across all the researchers. The main bases are represented by GATC. However, in certain experiments due to problems of sequencing the sequence identification is not clear at certain positions. In such cases if the ambiguity persists such that, for instance, the nucleotide cannot be differentiated as to whether it is G or C but one can be sure it is not A or T then the symbol used is S. Also since in most organisms DNA is double stranded one might face a problem if one starts encountering symbols that mean more than one base at a given position. In certain cases, the complimentary symbols are same as in the given strand because in both the cases they mean the same set of bases.
- How are the following bases represented?
a) guanine, (b) adenine, (c) Thymine, (d) Cytosine, (e) Purine, (f) Pyrimidine, (g) Amino, (h) Keto, (i) Strong Hydrogen bonds, (j) Weak Hydrogen bonds, (not-G, H follows G in the alphabet, (l) not-A, B follows a in the alphabet, (m) not-T (not-U), V follows U in the alphabet, (n) not-C, D follows C in the alphabet, (0) Any base.
G OR A
|Strong (3 Hydrogen bonds)
|Weak (2 Hydrogen bonds)
A C T
|Not-G, H follows G in the alphabet
G T C
|Not-A, B follows A in the alphabet
G C A
|Not-T (not-U),V follows U in the alphabet
G A T
|Not-C, D follows C in the alphabet
G A T C
- What kinds of analysis can be done using bioinformatics tools?
Many kind of analysis can be made using various bioinformatics tools. These are as follows:
a) Processing raw information- the bioinformatics tools are used to process the experimentally determined sequence/raw information into genes, the proteins encoded with their functions, the regulatory sequences, and inferring phylogenetic relationships.
b) Genes- Gene prediction can be done by using computer programmes like GeneMark for bacterial genomes and GENSCAN for eukaryotes.
c) Proteins- simple computer programmes are used to infer protein sequences from the predicted genes.
d) Regulatory sequences- Bioinformatics tools can be used to analyse and identify the regulatory sequences.
e) Inferring phylogenetic relationships- Information regarding the relationships between organisms can be obtained by aligning multiple sequences, calculating evolutionary distance, and constructing phylogenetic trees.
f) Making a Discovery- using the bioinformatics tools and databases, the functions of unknown genes can be predicted.
- What was the contribution of Margaret Dayhoff in the field of molecular evolution?
Margaret Dayhoff observed that protein sequences undergo variation during evolution according to certain patterns. She noted that:
- Amino acids were not replaced at random but were altered with specific preferences. For example, amino acids of similar physico-chemical characteristics were preferred, one over another.
- Some amino acids such as tryptophan, was generally not replaced by any other.
- Based on several homologous sequences, a point accepted mutation (PAM) matrix could be developed. This laid the first foundation for subsequent work on sequence comparisons using quantitative approaches.
- Who discovered the EST technique?
Craig Venter and his research group.
- What are IP and IPR?
With the fast pace development in the field of biotechnology, the issues related to legal characterization and the treatment of trade related biotechnological processes and products are of immense importance. These are popularly known as Intellectual Property. Intellectual Property includes Patents, trade secrets, copyrights, and trademarks. In biotechnology, the intellectual property covers the processes and products which result from the development of genetic engineering techniques through the use of restriction enzymes to create recombinant DNA.
Intellectual Property Rights (IPR) is a collective term applied to a number of different types of legal rights granted by each country. The right to protect this property prohibits others from making, copying, using or selling the proprietary subject matter.
- What are the advantages of getting a patent?
The advantages of patents and other forms of IPR are:
(h) These practices help in encouraging and safeguarding intellectual and artistic creations.
(i) They help to disseminate new ideas and technologies quickly and widely.
(j) They promote investment.
(k) They help the inventor to provide the fruits and benefits of his creation and invention to the public.
(l) They provide increased opportunities for the distribution of the above effects across countries in a manner proportionate to national levels of economic and industrial development (OECD, Paris, 1989)
- What are breeder’s right?
The new crop varieties are developed and protected through “plant breeder’s rights or PBRs. The PBRs ensures that the plant breeder who developed a particular variety gets the exclusive rights for marketing the variety. The following two major steps were taken in consideration of PBRs:
(b) The Food and Agriculture Organisation (FAO) has an International treaty on plant genetic resources for food and agriculture. This treaty consists of a particular clauses which refers to operation of farmer’s rights.
(c) The ‘Plant Varietal Protection and Farmer’s Rights Act 2001 agrees for the right of farmers, breeders, and researchers. The protection is provided by making compulsory licensing of rights, and inhibiting the import of plant varieties consisting of ‘genetic use of restriction technology’ (GURT) e.g. terminator technology of Monsanto.
Following conditions should be fulfilled to grant protection to the new varieties:
a) the new variety must always be new i.e. it should not have ever been exploited commercially.
b) It should be biologically distinct and possess different characters.
c) The new variety of the plant must have uniform characters.
f) The distinguishing character of new variety must be stable for generations.
g) The new variety should have taxonomic validity i.e. systematic position, generic and species names etc.
- What is the general layout of a patent?
A patent consists of three parts: the grant, specifications, and claims.
The Grant- The grant is filled at the patent office which is not published. It is the actual signed document which is the agreement that grants patent rights to the inventor.
Specifications- The specifications part is narrative and describes the subject matter and explains about the process of invention. It is published as a single document which is made public at a minimum charge from the patent office.
The claim- The claim section specifically defines the scope of the invention to be protected by the patent to which the others may not practice.
Reading a Patent
In order to file a patent, the documents required should have a specialized structure. An applicant should first file a patent in his or her own country and then at a later stage/date files in the international office. The application is prepared with a specific, clear and concise title. A patent attorney is appointed for the legal aspects of the patent whose job also involves filling the patent in the office of the Controller of Patents.
- Write down the full form of the following: (a) WIPO, (b) TRIPS, (c) GM, (d) PCT, (e) GURT.
(a) WIPO- World Intellectual Property Organisation, (b)TRIPS- Trade related Intellectual Property rights, (c) GM- Genetically modified, (d) PCT- Patent Co-operation Treaty, (e) GURT- Genetic use restriction technology.
- What do you mean by patenting live forms?
The EPO has suggested to patent the genetically engineered liveforms. “Onco mouse” is one of the examples of which initially the patent claims was rejected but on appeal the previous decision was overruled. Genetically engineered E.coli, in which human genes for insulin, growth hormone, t-PA etc have been introduced, have been patented in the USA. The transgenic herbicide and bollworm resistant cotton, and insect- resistant tobacco have been granted patents. Several countries such as Japan, USA, Europe, etc. have modified the patent laws stating that the transgenic plants and animals can be protected through patent claims.
- What are Trade secrets and Trademarks?
Trade secrets relate to private proprietary information or physical material that allows an advantage to the owner. Trade secrets in the biotechnology field includes materials like hybridization conditions, cell lines, corporate merchandise plans, customer lists etc. It is sometimes difficult to maintain trade secrets in biotechnology due to its large degree of research component.
If the trade secrets become public before the granted period, the intellectual is paid compensation and unauthorized users are punished by the Court.
The Trademark relates to distinctive words or symbols applied to products or services by manufacturers and helps to distinguish the goods of different companies. In biotechnology, the laboratory equipments, and some of the vectors useful in recombinant research are known by their trademarks. The public makes use of these trade works in order to choose whose goods they will have to buy. Trademark laws vary in every country and through agreement it is ensured that the trademark of one country must be protected in another country.
- What are the main areas of consideration for safety aspects in biotechnology?
The main areas of consideration for safety aspects in biotechnology are the following:
g) How to dispose off spent microbial biomass and purify the effluents from biotechnological processes?
h) The toxicity of the allergy associated with microbial production.
i) How to deal with the increase in the number of antibiotic resistant pathogenic microorganisms?
j) How to evaluate the pathogenicity of the genetically engineered microorganisms to infect humans, plants and animals?
- Write short notes on PCT and Budapest treaty.
The “Patent Co-operation Treaty”(PCT) was signed in 1970 and is administered by the World Intellectual Property Organization (WIPO) based in Geneva. Patent applications filed under PCTs are first processed by WIPO before being formally introduced into designated national systems. The patents granted by PCT are treated as International patent. The patent applications are processed by the WIPO which processes the patent application in an “International phase”. The international phase has concerns with: (a) formal preliminaries, (b) art search, (c) publication of patent application.
The Budapest Treaty (1980) provides for the recognition of culture collections at International Depository Authorities (IDA), in any of which a new strain of microorganism can be deposited for the purposes of a patent application in any member state. The strains which can serve the purpose of scientific community or society are accepted. These can be procured if required in future to work in Industry or research. The strains which are deposited are allocated an accession number, accession date, source of isolation and specific function. The purpose of convention of Budapest Treaty was to provide International Recognition of Microorganisms’ for the purpose of patent procedure.
- What is the role of copyright in biotechnology?
The copyright protection is only a form of expression of ideas. The patents and trade secrets provide protection of only basic know how, whereas copyrights protect the expressed materials viz. materials in printed, video-recorded r taped form. In the field of biotechnology, copyright usually may cover DNA sequence data which may be published. Computer data bases, photomicrographs of DNA instruction manuals can also be copyrighted.
- Fill in the blanks
a) ------------- is a government issued document that provides the holder the exclusive rights to manufacture, use or sell an invention for a defined period.
b)----------------- relates to work of literary or artistic craftsmanship, engineering drawings and software.
c)--------------- relates to distinctive words or symbols applied to products or services.
d) The structure of a patent has two parts--------------- and---------------.
(a) Patent, (b) Copyright, (c) Trademark, (d) Description, Claims
Sample question paper I
- Name two human diseases caused by the absence of a protein? (1)
- If a scientist is given a 200 Kb fragments of DNA to clone which vector will he prefer to use? (2)
- What is sparging? (1)
- Define “Subculturing”. (1)
- Which property of the plant cell is exploited to culture plant cells? (1)
- Give four advantages of Aqueous Two-Phase Partition Chromatography. (2)
- What is a Homologene? (1)
- A scientist needs to cut a DNA containing the palindromic sequence of GATC. Will he use a restriction endonulcease or DNase and why? (2)
- Why are antifoaming agents added in the microbiological cultures? (2)
- Give a few methods of concentration of protein. (3)
- Explain how embryo rescue can be used to produce novel hybrids. (3)
- Name two important products from animal cell culture technology. What are their
- How does the treatment with CaCl2 increase the competence levels of bacteria? (2)
- Suggest two methods of preserving microbial strains. (2)
- What do you man by term “Genomics”? (2)
- Indicate one application of site-directed mutagenesis. (2)
- What are the conventions adopted by the Database personnel to store nucleic acid data and protein sequence data with regard to the direction of the sequence? What is the basis of the convention? (3)
- A single E. Coli cell produces 10 molecules of a protein called repressor which has a molecular weight of 50,000 daltons. If the shape of the E. Coli cell resembles a cylinder of diameter 1 micrometer and height 2 micrometer, Calculate the number of E. Coli cells required as starting material to purify 1 g of the repressor if the purification yield is only 70%. (3)
- What is direct gene transfer? What are the methods of direct gene transfer? (5)
- Write a short note on protein based product. (5)
- Define the following: (a) Plasmid, (b) Restriction site, (c) Transformation, (d) Mutation, (e) Transfection (5)
- How can we use microbial cultures for the production of metabolites? (3)
- What is the possible benefit of embryonic stem cell technology? (3)
- What are the various measures taken to ensure protein stability during purification? (3)
- Write a short note on the bioethics in animal Genetic engineering. (5)
SAMPLE QUESTION PAPER II
- What is the car
bon part of BCAA used as? (1)
- What function does the enzymes DNA Ligase perform? (1)
- Under what conditions, continuous culture is most suitable? (1)
- What are the single letter IUPAC codes for alanine, glycine, tryptophan, tyrosine, serine, methionine? (3)
- Who proposed the concept of the famous “cell theory”? (1)
- Define: Subunit, domain, and quaternary structure in proteins. (3)
- What is molecular evolution? What are the observations regarding protein sequence variations vis-à-vis evolution? (3)
- What are the DNA probes? (2)
- What is the consequence if a protein is incorrectly folded? Give an example to illustrate your answer. (2)
- Give two examples of plants grown using callus culture. (1)
- What would happen if naked viral DNA is introduced into a bacterial cell? (2)
- What are the differences between Batch culture and Fed Batch culture? (3)
- Differentiate between functional and structural genomics? (3)
- What are the disadvantages of using E. Coli for production of eukaryotic proteins? (3)
- Differentiate between roller bottle and spinner cultures? (3)
- Recombinant insulin is produced at 100 mg/L by E. Coli at a cell concentration of 1 g/L. Calculate the volume of reactor (size of fermenter) needed for producing 1 kilogram of insulin in these conditions. (3)
- How frequently will a sequence GATC occur in the human genome
- What is BLAST and what are the principles involved in it? (3)
- Give the applications of the following: (a) Structural Proteomics, (b) Functional Proteomics, (c) Isoelectric Focussing, (d) Expression Proteomics, (e) Two-dimensional Gel Electrophoresis, (f) Edman Sequencing, and (g) Mass spectrometry.
- What are the conventions adopted by the Database personnel to store nucleic acid data and protein sequence data with regard to the direction of the sequence? What is the basis of the convention? (4)
- How can DNA cut with a staggered cutting enzyme joined (cloned)? (5)
- Write a short note on the history of bioinformatics?
- E. coli is a rod shaped bacteria about 2 micrometer long and 1 micrometer in diameter. The average density of a cell is 1.28 g/ml. Approximately 13.5% of the wet weight of E. coli is soluble protein. Estimate the number of molecules of a particular enzyme per cell if the enzyme has a molecular weight of 100,000 and represents 0.1% of the total soluble protein. (answer: 1626 molecules/cell) (5)
- Which types of dyes are used for DNA sequencing? (1)
- What is the use of Coulter counter? (1)