Cell Biology

Cell –

All living organisms are made up of cells. There are two types of cells, prokaryotes and eukaryotes. Prokaryotic cells have very little visible internal organization. They usually measure 1–2 μm across. Eukaryotic cells usually measure 10–100 μm across. They contain a variety of specialized internal organelles.

           Eukaryotic Plant Cell Eukaryotic animal cell

Structure and function of cell organelles –

Plasma Membrane :-

• Outermost layer in animal cells and in plant cells it is present just beneath the cell wall

• Made up of lipid bilayer – two layers of phospholipids embedded with proteins.
• separates the cell content from the external environment.
• Control selectively entrance & exit of materials.
• Plays major role in immune response.
• Involved in molecular recognition between cells.
• Contains enzymes and antigens

Nucleus –

Nucleus is consist of nuclear membrane, nucleoplasm, nucleolus and chromosomes. 

Nuclear membrane – It is a double layered structure encloses the content of the nucleus. Outer layer is connected to ER. Nucleus communicates with the remaining of the cell through several openings called nuclear pores. Nuclear pores are the site for exchange of large molecules between nucleus and cytoplasm. Nucleolus – dense, spherical structure present inside the nucleus. Plays an indirect role in protein synthesis by producing ribosomes. Chromosomes –  present in the form of strings of DNA  and histones called chromatin.

Endoplasmic Reticulum :-

it is a network of tubules and flattened sacs plays major role in production, processing and transport of proteins and lipids.

Two regions of the ER –

Rough ER – ribosomes are attached to the surface of the membrane. 

in leucocytes – produces antibodies

in pancreatic cells – produces insulin

Smooth ER – lacks attached ribosomes – involved in carbohydrates and lipid synthesis.           

In liver cells – production of enzymes to detoxify certain compounds.

In muscles – assist in contraction of muscle cell

In brain cells – synthesize male and female hormones

Golgi apparatus –

• Made up of series of flattened, stacked pouches called cisternae.
• Responsible for modifying, packaging and transporting of proteins and lipids into vesicles for delivery to targeted destinations.

Lysosomes –

Vesicular organelles form by breaking off from the Golgi apparatus. It Contain hydrolytic enzymes which allows the cell to digest –

1. Damaged cellular structures
2. Food particles ingested by the cell
3. Unwanted matter such as bacteria
4. Lysosomes are responsible of regression of tissues

Peroxisomes –

derived from smooth endoplasmic reticulum

• They contain oxidases to form hydrogen peroxides – highly oxidizing substance.
• Other oxidase enzymes present to oxidize substances that are poisonous to the cell.

Mitochondria –

self-replicating, double membraned structure.

• Outer membrane is smooth, inner membrane is folded into the cristae.
• The matrix of the mitochondria is a complex mixture of proteins and enzymes. These enzymes are important for the synthesis of ATP molecules, mitochondrial ribosomes, tRNAs and mitochondrial DNA.
• the cells with high need of energy have greater number of mitochondria.
• The liver cells mitochondria have enzymes that detoxify ammonia. 
• The mitochondria also play important role in the process of apoptosis or programmed cell death. 
• mt DNA is maternally inherited. enables to trace the maternal lineage.
• Mutations in the mitochondrial DNA leads to a number of illness like exercise intolerance.  

Chloroplast –

present in plant cells. Double membrane oval shaped organelle. Contains pigment chlorophyll

• site for photosynthesis.
• Possess its own DNA

Cytoskeletons –

Microfilaments & microtubules

Major structural components of several cell organelles including the asters, spindle and centriole

Other unique structures

• In human respiratory tract is lying with cells that have cilia
• Some bacteria have flagella for motility. Only human cells that has flagellum is sperm cells

Organization of cell –

Human body contains about 100 trillion cells. There are different types of cells in our body that organized into tissues to form different functional structure called as organs.

Different substances that make up the cell are called as protoplasm. Protoplasm composed of –

• Water
• Ions
• Proteins – structural & Functional
• Lipids
• Carbohydrates

Nucleic acids –

Nucleic acids are polynucleotides linked in a specific sequence by a phosphodiester bond. Two main types of nucleic acids are DNA and RNA. In DNA, nucleotide contains 2`- deoxyribose. In RNA nucleotide contains Ribose sugar.
Nucleotide – It is a nucleoside with one or two phosphate groups. Nucleoside – it is a compound consisting of a nitrogenous base covalently linked to pentose sugar

DNA – Deoxyribonucleic acid is made up of Phosphoric acid, a sugar molecule called deoxyribose and four nitrogenous bases (two purines, adenine and guanine, and two pyrimidines, thymine and cytosine)

DNA exists as a double helix (helix means spiral shaped) in most of the cells. Some viruses carry single stranded DNA. It is compacted into chromosomes by histones and other classes of proteins. The sequence of these four bases determines the nature of information. Every human cell carries 22 pairs of somatic chromosomes and one pair of sex chromosomes.

Structure of DNA:

DNA Replication –

The copying process in which a single DNA molecule becomes two identical molecules. On receiving signal for replication, chromatin becomes visible as a double strand, co-ordination of replicons on the chromosomes occur leading to replication of entire DNA. The strand of the original duplex (parent) separate and each individual strand serve as a template for the synthesis of new strand (replica).

The replica strands are synthesized by the addition of successive nucleotides by DNA polymerase enzyme in such a way that each base in the replica is complementary to the base across the way in the template strand.

It is a complex process with geometrical problems and requires variety of enzymes and other proteins.

Protein Synthesis- Central dogma

Transcription-

The process of making an RNA strand from DNA template is called as Transcription. Initiation of transcription occurs on chromatin template. Enzyme RNA polymerase interacts with promoter, transcription factor with enhancer which results into production of mRNAs, rRNAs and tRNAs.

Process of RNA spicing i.e removal of introns from pre- mRNA and joining of exons occurs resulting into mature mRNA. Only mature mRNA is translated to produce a protein molecule.

Translation–

Synthesis of polypeptide chain from mRNA is called Translation. The process takes place on ribosome. Base sequence in mRNA is translated in groups of three adjacent bases called as codons. Codons pairs with anticodon of aminoacyl tRNA and then the amino acid from aminoacyl tRNA gets transfer to growing polypeptide chain and tRNA is release.

The table of all codons and the amino acids they specify is called the genetic code

Protein folding and stability –

Protein folding is a complex process by which polypeptide chains attain a stable 3-dimensional structure through short range chemical interactions between nearby amino acids and long-range interactions between amino acids in different parts of the molecule. This process is facilitated by class of proteins known as chaperones.

During the folding process, the polypeptide chain twists and bends until it achieves a minimum energy state that maximize the stability of the resulting structure referred as native conformation.

We have briefly described the central dogma of molecular biology i.e

DNA makes DNA                         Replication

DNA makes RNA                         Transcript

DNA makes proteins                  Translation

A brief description of the functioning at cellular level may appear haphazard. Few definitions and bits of information and at the end the summary in three keywords! The fact is, the area of cell biology and molecular biology revolves around these three words. More we try to understand more we know the complexities of it.

Mutation –

Mutation is the process by which the sequence of base pairs in a DNA molecule is altered. A cell with a mutation is called as a mutant cell.

Mutation can occur in a number of ways, including through spontaneous changes, errors in the replication process (spontaneous mutation), or the action of radiation or particular chemicals (induced mutation).

An agent that induces mutation is called Mutagen. Many chemicals are not mutagenic themselves but are metabolized to mutagens (and carcinogens) in the body, often in the liver and other tissues. The Ames test is used for screening of potential mutagens.

Mutations could be simple such as substitution, insertion, deletion. Mutations can have range of effects. Some cause no detectable changes, others are lethal and still others have varying degree of effects.

By studying mutants that have defects in certain cellular processes, geneticists have made great progress in understanding how those processes take place. Various screening procedures have been developed to help find mutants of interest after mutagenizing cells or organisms.

Human genetic diseases resulting from DNA replication and repair mutation – Xeroderma pigmentosum, Ataxia telangiectasia, Fanconi anemia, Hereditary nonpolyposis colon cancer, etc.

DNA repair –

Effect of mutation can be reversed – Both prokaryotic and eukaryotic cells have a number of enzyme-based systems that repair DNA damage. If the repair systems cannot correct all the lesions, the result is a mutant cell (or organism) or, if too many mutations remain, death of the cell (or organism).

For e.g – DNA polymerase proofreading – During replication, when an incorrect nucleotide is inserted, the polymerase often detects the mismatched base pair and corrects the area by ―backspacing to remove the wrong nucleotide and then resuming synthesis in the forward direction.

Mutation in a gene can result in defective protein which can result in disease in the organism. (e.g. sickle cell anemia, cystic fibrosis, cancer, etc.)

Inborn Errors of Metabolism ( IEMs ) are a large group of rare genetic diseases that generally result from a defect in an enzyme or transport protein which results in a block in a metabolic pathway.

Inborne error of metabolism – e.g. Phenylketonuria (PKU)

If diagnosed soon enough after birth, patient can place on specially formulated diet low in phenylalanine.

Knowing the metabolic basis of the disease will eventually make it possible to develop methods for clinical intervention through diet, medication or other treatments that will ameliorate the severity of the disease.

Transposable elements –

Transposable elements are DNA segments that can insert themselves at one or more sites in a genome and can move to other sites in that genome. Transposable elements in a cell usually are detected by the changes they bring about in the expression and activities of the genes at or near the chromosomal sites into which they integrate. In bacteria, two important types of transposable elements are insertion sequence (IS) elements and transposons (Tn). Transposons also carry genes that encode other functions, such as drug resistance.

Genes and environment –

Most visible traits of organisms result from many genes acting together in combination with environmental factors. The relationship between genes and traits is often complex because

1. Every gene potentially affects many traits (pleiotropy), – children with extreme   forms of PKU often have blond hair and reduced body pigment 2. Every trait is potentially affected by many genes, and 3. Many traits are significantly affected by environmental factors as well as by genes.

Control of gene expression –

Regulation of gene expression is fundamental for the coordinate synthesis, assembly and localization of the macromolecular structures of cells. This is achieved by a multi-step program that is highly interconnected and regulated at diverse levels

1. Transcriptonal control of gene expression
2. Post- transcriptional control
3. Post translational control

Transcriptonal control –

Most of the genes are regulated at the first step in gene expression i.e the initiation of transcription. (DNA          RNA) DNA is transcribed into RNA by enzyme RNA polymerase.

In bacteria, RNA polymerase binds to the promoter sequence just upstream of the start site of transcription. The enzyme moves down the DNA template and synthesizes an RNA molecule. RNA synthesis stops once the enzyme has transcribed a terminator sequence. Bacterial genes encoding proteins for the same metabolic pathway are often clustered into operons. Some operons are induced in the presence of the substrate of their pathway, for example, the lac operon. Others are repressed in the presence of the product of the pathway, for example, the trp operon.

Eukaryotic mRNAs are modified by the addition of a 7-methyl-guanosine cap at their 5’ end. A poly-A tail is added to their 3’end. Intron sequences are removed, and the exon sequences joined together by the process known as splicing. The fully processed mRNA is then ready for transport to the cytoplasm and protein synthesis. In eukaryotes, there are three RNA polymerases—RNA polymerases I, II, and III. RNA polymerase II needs the help of the TATA-binding protein and other transcription factors to become bound to a promoter. This group of proteins is called the transcription preinitiation complex, and this is sufficient to make a small number of RNA molecules. However, to make a lot of RNA in response to a signal, such as a hormone, other proteins bind to sequences called enhancers. These proteins interact with the initiation complex and increase the rate of RNA synthesis.

Post Transcriptional control –

Control of gene expression at RNA level. Post-transcriptional regulation mechanisms comprise various processes such as mRNA processing (polyadenylation, capping and splicing), mRNA export and localization, mRNA decay, and mRNA translation.

Translational control –

The fate and location of proteins can be further controlled through modification of specific amino acids, cleavage by site-specific proteases and degradation through the proteasome.

Molecular Genetic Techniques

Genomics is the science of obtaining and analyzing the sequence of complete genome. Aspects of genomics and techniques used for genomic analysis –

DNA cloning – The production of many identical copies of a DNA molecule by replication in a suitable host. Steps in DNA cloning –

1. Isolate DNA from an organism.
2. Cut the DNA into pieces with a restriction enzyme – an enzyme that recognizes and cuts within a specific DNA sequence—and insert (ligate) each piece individually into a cloning vector cut with the same restriction enzyme to make a recombinant DNA molecule, a DNA molecule constructed in vitro containing sequences from two or more distinct DNA molecules.
3. Introduce (transform) the recombinant DNA molecules into a host such as E. coli. The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA.
4. The vector DNA is isolated (or separated) from the host cells’ DNA and purified. 

Several types of vectors have been constructed for cloning DNA like plasmids, bacteriophages, cosmids, and artificial chromosomes. The vector types differ in their molecular properties and in the maximum amount of inserted DNA they can hold. The cloned DNA can be used to:

• Work out the function of the gene
• Investigate a gene’s characteristics (size, expression, tissue distribution)
• Look at how mutations may affect a gene’s function
• Make large concentrations of the protein coded for by the gene. (for eg insulin)

Gel Electrophoresis – it is a technique used to separate DNA fragments produced by restriction enzyme on the basis of their size and charge.it is necessary to select the fragments that are the right size for cloning in the vector being used, and to eliminate those that are either too small or too large.

Nucleic Acid Hybridization – Denatured DNA strands can, under certain conditions, form double-stranded DNA with other strands, provided that the strands are sufficiently complementary in sequence. This process of renaturation is called nucleic acid hybridizationbecause the double-stranded molecules are “hybrid” in that each strand comes from a different source. Hybridization of a labeled nucleic acid to complementary sequences can identify specific nucleic acids.

Polymerase Chain Reaction (PCR) – it is a common laboratory technique used to amplify DNA segments in vitro.

RT –PCR: Reverse Transcription PCR – RNA is first converted to cDNA and then the cDNA is amplified by PCR. RT-PCR is a very sensitive technique for detecting and quantifying RNA, often mRNA.

Real-time PCR (real-time quantitative PCR) is a PCR method for measuring the increase in the amount of DNA as it is amplified. Real-time PCR is used extensively to quantify mRNA levels for many genes in a wide range of cells and tissues in many organisms. For example, real-time PCR is used diagnostically to detect HIV and hepatitis C virus.

DNA microarrays: monitor the expression of thousands of genes simultaneously. Application:

1. To know which genes are active in a cell at one time vs another, or before and after some treatment
2. How active are the genes?
3. Does the genome of one organism contain genes similar to those in the genome of another?
4. Useful for screening for genetic diseases, including cancers

Applications of molecular techniques:

1. Site specific mutagenesis of DNA
2. Analysis of expression of individual genes
3. Analysis of protein-protein interactions

Gene therapy –

Two types of gene therapy are possible:

1. somatic cell therapy – in which somatic cells are modified genetically to prevent a genetic defect in the individual receiving the therapy;
2. germ-line cell therapy – in which germ-line cells are modified to correct a genetic defect.

Somatic cell therapy results in a treatment for the genetic disease in the individual, but progeny could still inherit the mutant gene. Germ-line cell therapy, however, could prevent the disease because the mutant gene can be replaced by the normal gene and that normal gene would be inherited by the offspring. Both somatic cell therapy and germ-line cell therapy have been used successfully in nonhuman organisms, including mice, but only somatic cell therapy has been used in humans because of ethical issues raised by germ-line cell therapy.

Evolutionary Genetics –

The process of evolution essentially consists of mutation and selection. Following diagram illustrates the steps which may differ drastically in mutations and subsequent functions.

Viruses, plasmids, transposons, and retrotransposons –

Viruses are mobile infectious genetic material. They are incapable of growth and replication without the aid of host cell. The origin of viruses is unknown. Viruses can have either RNA or DNA bases genome.

Transposons are mobile genetic elements that have ability to move from a location of a genome to another. Some move as DNA element (transposons) while others as RNA intermediates (retrotransposons)

Plasmids are autonomously replicating DNAs that are common in bacteria but are found in many other organisms and organelles.

Many types of viruses, plasmids, transposons and introns are related and are members of a large number of mobile genetic elements. Mobile genetic elements can cause no. of deleterious as well as useful changes.

Multigene Families –

It is believed that some of the first genes might have taken long time to evolve further. For faster evolution gene duplication followed by mutations and divergence might have occurred. The presence of similar regions in very different genes and proteins are indicative of it.

One important aspect of multigene family is that orthologues and paralogues versions of the genes can be formed. It means, if the gene gets evolved in many different species and its protein product retains the same function in all, each of those genes in each of those species is orthologues. However, if the copy of the gene and its product are quite different than the original, they are paralogues.

Many important evolutionary processes can be attributed to multigene families. Some of the complex structures and systems in organisms are  the  result  of  evolution  of multigene families.