Interphase precedes both mitosis and meiosis and is the period between cell divisions during which time the chromosomes replicate and the chromosomes are not visible (loosely packed). During interphase, two pairs of centrioles lie next to each other, just outside the nucleus.

Mitosis is a process where in, one parent cell gives rise to two identical daughter cells. Mitosis can be divided into four stages: Prophase, Metaphase, Anaphase and Telophase.

Prophase: Chromosomes (two identical copies) condense, each chromosome has two arms and each copy of chromosome is called Chromatid. Spindle fibers form at centriole and centriole begin to separate. In addition, nuclear membrane disappears.

A short period just before metaphase, called prometaphase, comprises movement of centrioles to opposite ends of the cell and attachment of spindle fibers to each of the chromatids.

Metaphase: Chromosomes line up along an imaginary line, called the metaphase plate that divides the cell into two. The spindle fibers begin to pull the chromosomes to the opposite ends of the cell.

Anaphase: Spindle fibers separate sister chromatids to opposite ends of the cell.

Telophase: Chromatids, now called chromosomes move to each pole and new nuclear membranes form.

Once mitosis is complete, the rest of the cell divides, by a process called cytokinesis (division of the cytoplasm) and cell division is complete.

Meiosis is a type of cell division that is specific to reproduction and results in 4 daughter cells that have half the number of unidentical chromosomes (genetic information is contained from both parents). Meiosis is divided into two phases: Meiosis I and Meiosis II.

Meiosis I: comprises Prophase I, Metaphase I, Anaphase I and Telophase I

Prophase I: Chromosomes attach to nuclear membrane and pair up with corresponding chromosome (to from a tetrad) from the other parent. Homologous recombination occurs between chromosome pairs and genetic material exchange takes place.

Prometaphase I: Similar to prometaphase I in mitosis except, one chromosome (instead of chromatid) from the homologous pair is attached to each centriole. Therefore, 23 chromosomes (in humans) attach to fibers from one centriole and remaining 23 attach to the fibers from the other centriole.

Metaphase I: Chromosome pairs line up along the metaphase plate on either side.

Anaphase I: Chromosome pairs separate. One half of the chromosomes goes to one pole and the other half to the other pole.

Telophase I: Chromosomes reach opposite ends of the cell and a nuclear membrane forms marking the end of Meiosis I.

There is a major distinction between sperm and egg cells at this stage. While in sperm cells the cytoplasm is equally divided between the two emerging daughter cells, in oocytes, the cytoplasm is concentrated in one of the emerging daughter cells resulting in a large and a small daughter cell called the polar body.

Telophase I is followed by cytokinesis resulting in two daughter cells in case of sperms and one large cell and one small cell (polar body) in the case of the egg (primary oocyte to be precise).

Meiosis II follows a very short Interphase II but chromosome replication does not take place unlike in Mitosis and Meiosis I.

Meiosis II can also be divided into four phases: Prophase II, Metaphase II, Anaphase II and Telophase II. Meiosis II is very similar to Mitosis

Prophase II: Chromosomes condense, spindles form centrioles begin to separate and the nuclear membrane disappears. There is no homologous recombination.

Prometaphase II: Spindle fibers attach to chromatids and centrioles move to opposite ends of cell.

Metaphase II: Chromosomes align along the metaphase plate and fibers begin to pull at the chromosomes.

Anaphase II: Sister chromatids are pulled apart toward opposite ends of the cells.

Telophase II: Chromatids arrive at opposite poles, nuclear membranes form. Again, as in Telophase I, in the female cell, the emerging daughter cells will have unequal distribution of the cytoplasm resulting in one large and another small cell. The resulting large cell becomes the egg or ovum and the smaller cell is called the polar body. The first polar body formed at the end of Meiosis I also divides to form two polar bodies. Therefore, in females, at the end of Meiosis, there is one egg cell and three polar bodies.

Cytokinesis follows Telophase II to mark the completion of cell division.

Main differences between Mitosis and Meiosis I:

Prophase

Mitosis: Chromatids of chromosome begin to separate. There is no exchange of any genetic material
Meiosis I: Pairing of homologous chromosomes, tetrad formation and homologous recombination (exchange of genetic material) take place

Metaphase

Mitosis: Chromosomes line up along metaphase plate
Meiosis I: Chromosome pairs line up along metaphase plate

Anaphase

Mitosis: Sister chromatids pulled to opposite ends of cell
Meiosis I: Separation of chromosome pairs to opposite ends of cell

Telophase and Cytokinesis

Mitosis: Two daughter cells with identical chromosomes and exact number of chromosomes as parent cells
Meiosis I: Two daughter cells with chromosomes from both parents and half the number as parent cells and this is followed by Meiosis II

PS: Opposite ends of cell and opposite poles have been used interchangeably

Cells of the Immune System: derived from the hematopoietic stem cell

1. Lymphoid Lineage

T lymphocytes (T cells, made in the thymus)

B lymphocytes (B cells, made directly from the bone marrow)

Natural Killer cells (NK cells)

2. Myeloid lineage

Monocytes that give rise to macrophages

Langerhans cells and Dendritic cells

Megakaryocytes that give rise to Platelets

Granulocytes (eosinophils, basophils and neutrophils)

Primary lymphoid tissues: bone marrow and thymus

Secondary lymphoid tissues: spleen, lymph nodes

Leukocyte migration: T and B cells leave the thymus and bone marrow respectively as naïve lymphocytes, migrate into the blood and then into the secondary lymphoid tissue. Antigen presenting cells (APCs), such as dendritic cells, also derived from the bone marrow, migrate into tissues, take up antigen and bring it back to the secondary lymphoid tissues to present the antigen to the T and B cells. The T and B cells are now primed or activated and they migrate to the sites of infection and inflammation to mount an attack.

Immune Response

Pathogens usually have two locations: Extracellular and Intracellular

Extracellular Pathogens are targeted by antibodies by at least one of three processes: Neutralization, Opsonization and Complement Activation

Neutralization: antibody may bind to bacterial toxin and neutralize, thereby preventing the pathogen from interacting with host cells. These antibody tagged toxins are later degraded

Opsonization: Antigens are coated with antibodies and are targeted for phagocytosis.

Complement Activation: Antibodies coat bacterial cells and these antibodies act as receptors for the first protein of the complement system, eventually forming a protein complex leading usually to phagocytosis.

Antibodies in each class have different sites of action and therefore vary in their effectiveness in neutralization, opsonization and complement activation.

Intracellular Pathogens are targeted by a T-cell mediated response. There are two intracellular locations:

Cytosol (continuous with nucleus via nuclear pore): site of all viruses and some bacteria

Vesicular System (ER, Golgi, endosomes, lysosomes etc): site of some bacteria and some parasites

There are also two T cells and the intracellular location determines the type of T cell.

Cytotoxic T cells (Tc or CTL): Express CD8 and kill pathogens in cytosol

Helper T cells (Th): Express CD4 and are again of two kinds

Inflammatory Th1 that kill vesicular pathogens

Th2 (True helper cells) are involved in antibody production by B cells against T-dependent antigens on extracellular pathogens.

Both antibody (humoral) and cell-mediated responses contribute to eliminating the pathogen.

Note: this explanation is best understood if you have a Lineweaver-Burke plot and a velocity vs. concentration graph available while reading it.

When you look at an enzyme reaction, you’re really looking at two reactions:

E+S <–> ES –> E + P

So in those rxns, E is the enzyme, S is the substrate, and P is the product. You’ll notice that substrate binding is reversible, so you could say that we’re looking at three possible reactions. Call the first forward reaction R1. Call the reverse of that reaction R2. Call the release of product from the enzyme R3.

So what happens with the Michaelis constant, Km, is that you make a ratio out of the rates of those three reactions to come up with a ratio for the overall reaction. That ratio is (R2+R3)/R1. So take a look at that ratio, and think about this: R2 and R3 are the two reactions that remove the substrate from the enzyme, and R1 is the reaction that binds the substrate to the enzyme. This means that Km is a ratio of separation:binding. So Km is related to the affinity of the enzyme for the substrate.

Now look at the velocity vs. concentration curve. Km is the substrate concentration at 1/2 of Vmax. Remember that Vmax is the mechanical limit of the enzyme — it’s churning out the product as fast as it possibly can. So look at a pair of enzymes, one with a high Km, and one with a low Km. An enzyme with a low Km reaches 1/2 Vmax at very low concentrations, because the enzyme has a high affinity for the substrate. An enzyme with a high Km, though, doesn’t have a strong affinity for the substrate, so it takes a lot more of the substrate to get the enzyme up to 1/2 Vmax.

Now look at the Lineweaver-Burke plot of 1/Vo vs. 1/[substrate], aka the double reciprocal plot. The important things to remember about Lineweaver-Burke plots are the x and y intercepts. The x-intercept = -1/Km, and the y-intercept = 1/Vmax. Just learn these, and I’ll help you make sense of them by discussing inhibition.

InhibitionThe best way to understand these graphs is to look at what happens with different types of inhibition.

First, think about competitive inhibition. You’ve got another substrate competing for the same enzyme. So what changes? Well, the enzyme suddenly has something else it can bind to, so its affinity for the substrate is reduced. At the same time, if you cram in enough substrate to overwhelm the competition, you can eventually reach Vmax. So in competitive inhibition, Km increases while Vmax remains the same. Look at your V/[s] graph, and the curve will stretch, because it takes a lot more substrate to get that Km at 1/2 Vmax. Look at your Lineweaver-Burke plot. The y-intercept stays the same because Vmax doesn’t change. But Km has gone up, which means that -1/Km has gotten closer to zero, increasing the slope of the line and rotating it on the y-axis.

Now look at noncompetitive inhibition. In noncompetitive inhibition, you have something binding to another site on the enzyme, changing the structure of the binding site, and thus affecting the amount of enzyme that is able to bind substrate. This means that Vmax is reduced. Km, the affinity of the functional enzyme, remains the same, though. Looking at the V/[s] curve, you simply squish the maximum down. Looking at Lineweaver-Burke, Km is the same, so your x-intercept doesn’t move. Vmax is smaller, so 1/Vmax is larger. This means that your line will have a higher slope and rotate on the x axis.

There are other conditions possible, but that covers the basics. Oh, and notice I didn’t actually mention the Michaelis-Menten equation or the Lineweaver-Burke equation. Questions involving those are memorization w/plug & chug calculation. Understanding what happens on the graphs is much more intuitive.

Please feel free to add if I may have missed anything or correct if I’m even partially wrong.

tRNA molecule
• Single chain, contains 73-93 ribonucleotides
• Contains many unusual bases such as inosine, pseudouridine
• tRNA is L shaped
• 5’ end is phosphorylated
• 3’ end ends in CCA and contains the amino acid attachment, it is at one end of the L
• The other end of the L, far from the amino acid end, is the anticodon loop

The process of translation, like transcription, is also divided into three phases:
initiation, elongation and termination. These three phases are regulated by initiation, elongation and termination factors respectively.

Initiation

Initiator tRNA (tRNAi) that carries methionine is the only tRNA capable of initiating translation. An initiation complex called 43S, comprising methionine tRNAi, the small 40S ribosomal subunit, and initiation factors such as eIF2. The 43S complex is recruited to the 5’ end of the mRNA by eIF4E. This complex now scans the mRNA in the 5’ to 3’ direction to find the first 5’-AUG-3’. Scanning is an ATP dependent process. As soon as the met-tRNAi finds the first AUG, the larger ribosomal subunit is recruited and this recruitment is mediated by eIF5. Assembly of the large ribosomal subunit completes the initiation step. The large subunit has 3 binding sites, E, P and A and the first codon (AUG) is aligned at the P site.

Elongation

Elongation begins with the delivery of an amino-acyl tRNA (corresponding to the appropriate codon on the mRNA) to the A site on the ribosome by EF-Tu and this is followed by GTP hydrolysis. A peptide bond, catalyzed by peptidyl transferase, is formed between methionine and the aminoacyl tRNA by the transfer of methionine to the A site, leaving the deacylated tRNA at the P site. The next step of elongation is translocation, where, the deacylated tRNA moves to the E site, the dipeptidyl-tRNA (met + aminoacyl tRNA) moves to the P site and the mRNA moves forward by 3 bases, thereby aligning the next codon for the appropriate aminoacyl tRNA. Translocation is mediated by elongation factor G. A and E sites cannot be occupied at the same time, therefore, as soon as the A site is occupied, the E site containing the deacylated tRNA is emptied. Elongation proceeds in this fashion until a stop codon is encountered.

Termination

Normally, tRNAs do not have anticodons corresponding to the stop codons (UAA, UAG or UGA. At termination the polypeptide chain is at the P site and the stop codon is at the A site. Stop codons are recognized by proteins called release factors (RFs) or termination factors. Peptidyl transferase is activated when an RF binds to a termination codon at the A site. The activated peptidyl transferase hydrolyzes the bonds between the polypeptide and the tRNA at the P site. The released polypeptide chain, tRNA and mRNA leave the ribosome in that order. The ribosome dissociates into its subunits ready for another round of protein synthesis.

Summarizing eukaryotic protein translation

• mRNA is always translated in the 5’ to 3’ direction
• proteins are synthesized in the amino to carboxyl direction
• several ribosomes can simultaneously translate an mRNA molecule and such an mRNA molecule (with many ribosomes attached) is called a polysome or a polyribosome
• amino acids are added sequentially to the carboxyl end of a polypeptide chain
• aminoacyl tRNAs are the activated precursors in which the carboxyl group of an amino acid is attached to the 3’ hydroxyl group of a tRNA
• the above step is catalyzed by an aminoacyl tRNA synthetase and is driven by ATP
• initiator tRNA, met-tRNAi, occupies peptidyl (P) site, the next aminoacyl tRNA, added during elongation, occupies the aminoacyl (A) site
• peptide bond is formed between carboxyl group of met and aminoacyl tRNA
• dipeptidyl tRNA moves from A to P site
• deacylated tRNAi moves to E (exit) site and leaves ribosome
• a new aminoacyl tRNA occupies A site
• elongation proceeds until stop codon is encountered
• stop codon (UGA, UAA, or UAG) recognized by release factors that facilitate release of the completed polypeptide from the ribosome