Biosynthesis of fatty acids and phospholipids

Biosynthesis of fatty acids and phospholipidsBy : Matt Daniel M. Daep

Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and NADPH through the action of enzymes called fatty acid synthases. This process takes place in the cytoplasm of the cell. Most of the acetyl-CoA which is converted into fatty acids is derived from carbohydrates via the glycolytic pathway. The glycolytic pathway also provides the glycerol with which three fatty acids can combine (by means of ester bonds) to form triglycerides (also known as "triacylglycerols", to distinguish them from fatty "acids" - or simply as "fat"), the final product of the lipogenic processWhat is fatty acid synthesis:

The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonyl-CoA.The ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid. The spontaneous decarboxylation drives the condensation. Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA.As with other carboxylation reactions (e.g., Pyruvate Carboxylase), the enzyme prosthetic group is biotin.

Phospholipids are a class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic. The structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are joined together by a glycerol molecule. The phosphate groups can be modified with simple organic molecules such as choline.

The first phospholipid identified in 1847 as such in biological tissues was lecithin, or phosphatidylcholine, in the egg yolk of chickens by the French chemist and pharmacist, Theodore Nicolas Gobley. Biological membranes in eukaryotes also contain another class of lipid, sterol, interspersed among the phospholipids and together they provide membrane fluidity and mechanical strength. Purified phospholipids are produced commercially and have found applications in nanotechnology and materials science.What are phospholipids?

Phosphatidic Acid

Biosynthesis:

While quantitatively a minor component of membrane phospholipids, phosphatidic acid forms the backbone on which the synthesis of other phospholipid species and triacylglycerol is based.Biosynthesis

Phosphatidic acid synthesis begins with the addition of a fatty acyl-CoA, usually saturated, to glycerol 3-phosphate at the sn-1 position to produce lysophosphatidic acid. This reaction is catalyzed by glycerol 3-phosphate acyltransferase and is rate limiting for phosphatidic acid synthesis. There are two forms of this enzyme; one is found in the outer mitochondrial membrane, while the other is found in the endoplasmic reticulum. A second fatty acyl-CoA, often unsaturated, is added to lysophosphatidic acid at the sn-2 position by acylglycerol-3-acyltransferase to form phosphatidic acid. This occurs primarily in the endoplasmic reticulum.

Biosynthesis of ketones, sterols, isoprenoids

Ketone bodies are three water-soluble molecules (acetoacetate, beta-hydroxybutyrate, and their spontaneous breakdown product, acetone) that are produced by the liver from fatty acids[1] during periods of low food intake (fasting), carbohydrate restrictive diets, starvation, prolonged intense exercise,[2] or in untreated (or inadequately treated) type 1 diabetes mellitus. These ketone bodies are readily picked up by the extra-hepatic tissues, and converted into acetyl-CoA which then enters the citric acid cycle and is oxidized in the mitochondria for energy.[3] In the brain, ketone bodies are also used to make acetyl-CoA into long-chain fatty acids.What are ketones?

Ketone bodies are produced by the liver under the circumstances listed above (i.e. fasting, starving, low carbohydrate diets, prolonged exercise and untreated type 1 diabetes mellitus) as a result of intense gluconeogenesis, which is the production of glucose from non-carbohydrate sources (not including fatty acids). They are therefore always released into the blood by the liver together with newly produced glucose, after the liver glycogen stores have been depleted. (These glycogen stores are depleted after only 24 hours of fasting.)Ketone synthesis

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones.Isoprenoids

Isoprenoid quinones are membrane-bound compounds found in nearly all living organisms. The only exception presently known is some obligatory fermentative bacteria that lost the ability of synthesis of isoprenoid quinones and methanogenic Archea, belonging to Methanosarcinales . Isoprenoid quinones are composed of a polar head group and a hydrophobic side chain. The apolar isoprenoid side chain gives the molecules a lipid-soluble character and anchors them in membrane lipid bilayers, whereas the hydrophilic head group enables interaction with hydrophilic parts of proteins. It is generally accepted that long-chain, isoprenoid quinones localize in the hydrophobic mid-plane region of the lipid bilayer, whereas the polar head can oscillate between mid-plane and polar interphase of the membrane . The quinone ring can undergo two-step reversible reduction leading to quinol form . The reduced form of isoprenoid quinones is more polar and the quinol head group is thought to preferentially localize in polar, interphase region of membranes and.Synthesis

Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells.What are sterols

Figure 1: Molecular structures of cholesterol and ergosterol. The arrows indicate the parts of the molecules which have been shown to be essential for the growth of mammalian cells (cholesterol), fungi, and trypanosomatids (ergosterol and 24-methyl sterols).

Figure 1:

Figure 2: Schematic representation of main morphologies found during the life cycle of some members of the Trypanosomatidae family in the invertebrate host (insect) and vertebrate host (mammal).

Figure 2:

Figure 3: The biosynthesis of ergosterol and cholesterol showing the main steps, the enzymes involved, and the known inhibitors.

Figure 3

Cholesterol regulation and Lipoproteins

A compound of the sterol type found in most body tissues. Cholesterol and its derivatives are important constituents of cell membranes and precursors of other steroid compounds, but a high proportion in the blood of low-density lipoprotein (which transports cholesterol to the tissues) is associated with an increased risk of coronary heart disease.What is cholesterol?

The amount of cholesterol that is synthesized in the liver is tightly regulated by dietary cholesterol levels. When dietary intake of cholesterol is high, synthesis is decreased and when dietary intake is low, synthesis is increased. However, cholesterol produced in other tissues is under no such feedback control. Cholesterol and similar oxysterols act as regulatory molecules to maintain healthy levels of cholesterol.Cholesterol regulation

The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5 side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the amino-terminal domain is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription. When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways.

any of a group of soluble proteins that combine with and transport fat or other lipids in the blood plasma.What are lipoproteins?

LPL actions within tissues are modulated at both the transcriptional and posttranscriptional levels. The latter might involve actions of the glycosylphosphatidylinositol HDL binding protein (GPIHBP) protein (19), angiopoietin-like proteins, which reduce LPL dimer formation (20), and the recently described lipase maturation factor (21). LPL regulation is tissue specific. LPL is present in the liver during fetal and early postnatal life but is then suppressed by a putative transcriptional regulatory mechanism, perhaps involving a novel transcription factor, termed RF-1-LPL, which binds to an NF-1-like site in the region of the glucocorticoid response element. This extinction of the hepatic expression of LPL is also under the influence of thyroid hormone and glucocorticoids.

Amino acid synthesis is the set of biochemical processes (metabolic pathways) by which the various amino acids are produced from other compounds. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesise all amino acids. Humans are excellent example of this, since humans can only synthesise 11 of the 20 standard amino acids (aka non-essential amino acid), and in time of accelerated growth, histidine, can be considered an essential amino acid.What is amino acids synthesis?

Most amino acids are synthesized from -ketoacids, and later transaminated from another amino acid, usually glutamate. The enzyme involved in this reaction is an aminotransferase.

-ketoacid + glutamate amino acid + -ketoglutarateGlutamate itself is formed by amination of -ketoglutarate:

-ketoglutarate + NH+4 glutamate

Porphyrins are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their carbon atoms via methine bridges (=CH). The parent porphyrin is porphin, and substituted porphines are called porphyrins. The porphyrin ring structure is aromatic, with a total of 26 electrons in the conjugated system. Various analyses indicate that not all atoms of the ring are involved equally in the conjugation or that the molecule's overall nature is substantially based on several smaller conjugated systems. One result of the large conjugated system is that porphyrin molecules typically have very intense absorption bands in the visible region and may be deeply colored; the name "porphyrin" comes from the Greek word (porphyra), meaning purple.Porphyrins

Illustration:

Biosynthesis of nucleotides

Nucleotides are small organic molecules consisting of a five ring sugar (which can be a ribose or a deoxyribose), a nitrogen base, and one to three phosphate groups. The most important function of nucleotides is there polymerization in nucleic acids such as DNA or RNA. As such they serve as the building blocks for the extremely long molecules that make up the chromosomes in every cell of our bodies, and that carry the genetic blueprint. The genetic information is carried out in DNA, and is organised in genes. Messenger RNA, is transcribed from the DNA whenever genes are expressed and carries the information from the cell nucleus to the cytoplasm, where it acts as a blueprint for protein synthesis.

What are nucleotides?

Nucleotides are required for cell growth and replication

A key enzyme for the synthesis of one nucleotide is dihydrofolate reductase. Cells grown in the presence of methotrexate, a reductase inhibitor, respond by increasing the number of copies of the reductase gene. The bright yellow regions visible on three of the chromosomes in the fluorescence micrograph (left), which were grown in the presence of methotrexate, contain hundreds of copies of the reductase geneNucleotide synthesis

Da en

DNA and Chromosome Structure

DNA (or deoxyribonucleic acid)is the molecule that carries the genetic information in all cellular forms of life and some viruses. It belongs to a class of molecules called the nucleic acids, which are polynucleotides - that is, long chains of nucleotides.Each nucleotide consists of three components:---a nitrogenous base: cytosine (C), guanine (G), adenine (A) or thymine (T)---a five-carbon sugar molecule (deoxyribose in the case of DNA)---a phosphate molecule

The backbone of the polynucleotide is a chain of sugar and phosphate molecules. Each of the sugar groups in this sugar-phosphate backbone is linked to one of the four nitrogenous bases.Strand of polynucleotides

Strand of polynucleotides

DNA's ability to store - and transmit - information lies in the fact that it consists of two polynucleotide strands that twist around each other to form a double-stranded helix. The bases link across the two strands in a specific manner using hydrogen bonds: cytosine (C) pairs with guanine (G), and adenine (A) pairs with thymine (T).

Double strand of polynucleotides

The double helix of the complete DNA molecule resembles a spiral staircase, with two sugar phosphate backbones and the paired bases in the centre of the helix. This structure explains two of the most important properties of the molecule. First, it can be copied or 'replicated', as each strand can act as a template for the generation of the complementary strand. Second, it can store information in the linear sequence of the nucleotides along each strand.

ChromosomesEukaryotic chromosomes

The labeleukaryoteis taken from the Greek for 'true nucleus', and eukaryotes (all organisms except viruses, Eubacteria and Archaea) are defined by the possession of a nucleus and other membrane-bound cell organelles.The nucleus of each cell in our bodies contains approximately 1.8 metres of DNA in total, although each strand is less than one millionth of a centimetre thick. This DNA is tightly packed into structures calledchromosomes, which consist of long chains of DNA and associated proteins.

In eukaryotes, DNA molecules are tightly wound around proteins - calledhistone proteins- which provide structural support and play a role in controlling the activities of the genes.A strand 150 to 200 nucleotides long is wrapped twice around a core of eight histone proteins to form a structure called a nucleosome. The histone octamer at the centre of thenucleosomeis formed from two units each of histones H2A, H2B, H3, and H4. The chains of histones are coiled in turn to form asolenoid, which is stabilised by the histone H1. Further coiling of the solenoids forms the structure of the chromosome proper.

Each chromosome has ap armand aq arm. The p arm (from the French word 'petit', meaning small) is the short arm, and the q arm (the next letter in the alphabet) is the long arm. In their replicated form, each chromosome consists of twochromatids.

Chromosome unraveling to show the base pairings of the DNAThe chromosomes - and the DNA they contain - are copied as part of the cell cycle, and passed to daughter cells through the processes of mitosis and meiosis.

Human beings have 46 chromosomes, consisting of 22 pairs ofautosomesand a pair ofsex chromosomes: two X sex chromosomes for females (XX) and an X and Y sex chromosome for males (XY). One member of each pair of chromosomes comes from the mother (through the egg cell); one member of each pair comes from the father (through the sperm cell).A photograph of the chromosomes in a cell is known as akaryotype. The autosomes are numbered 1-22 in decreasing size order.

Karyotype of a human male

Theprokaryotes(Greek for 'before nucleus' - including Eubacteria and Archaea) lack a discrete nucleus, and the chromosomes of prokaryotic cells are not enclosed by a separate membrane.Prokaryotic chromosomesMost bacteria contain a single, circular chromosome. (There are exceptions: some bacteria - for example, the genus Streptomyces - possess linear chromosomes, and Vibrio cholerae, the causative agent of cholera, has two circular chromosomes.) The chromosome - together with ribosomes and proteins associated with gene expression - is located in a region of the cell cytoplasm known as thenucleoid.

In addition to the main chromosome, bacteria are also characterised by the presence of extra-chromosomal genetic elements calledplasmids. These relatively small circular DNA molecules usually contain genes that are not essential to growth or reproduction.In addition to the main chromosome, bacteria are also characterised by the presence of extra-chromosomal genetic elements calledplasmids. These relatively small circular DNA molecules usually contain genes that are not essential to growth or reproduction.Retrieved from http://www2.le.ac.uk/departments/genetics/vgec/schoolscolleges/topics/dna-genes-chromosomes

DNA Replication

Every time a cell divides to produce new cells its DNA is copied. Each molecule of DNA undergoes semi-conservative replication. Put very simply, the DNA unwinds and unzips to expose nucleotide bases. DNA polymerases catalyse the addition of activated DNA nucleotides, according to complementary base-pairing rules, to make two new identical molecules of DNA, each one containing one old strand and one new strand. Hence each new molecule contains half of the original moleculeBefore DNA synthesis begins the original strands are separated and the synthesis of the daughter strands begins at the replication fork at a site called an origin of replication where a replisome is assembled from many proteins. The initiation complex that is formed attracts DNA polymerases.Synthesis of the new strands is called elongation and is aided by the proteins in the replisome.

Lastly the termination site replicates

Figure 1. The DNA replication fork. Because both daughter strands are synthesised in the 5 to 3 direction, the DNA complementary to the lagging strand is synthesised in small fragments called Okazaki fragments. These fragments are then joined together.

Figure 2. Enzymes involved in DNA replication.

The replisomeThe replisome consists of many proteins, including helicase, gyrase/ topoisomerase, primase, DNA polymerases, RNAse H and ligase. One DNA polymerase complex synthesises the lagging strand and another synthesises the leading strand. There are also factors, called replication proteins, that protect both the unstable single-stranded unwound leading and lagging strands from making hydrogen bonds with themselves and forming hairpins.Helicase causes the hydrogen bonds between complementary base pairs to break and so catalyses the separation of the two parental strands that will act as templates for synthesis of the daughter molecules. Helicase moves along the DNA in a 3 to 5 direction.Helicase

Gyrase (a form of topoisomerase) unwinds the resulting supercoil that forms upstream of the section of unwound DNA.GyraseDNA polymerases catalyse the elongation phase of replication.DNA polymerases Clamp proteins help keep the DNA polymerases attached to the leading and lagging strands and make sure the process proceeds at a suitably fast rate.Clamp proteins

PrimingIn eukaryotic cells a DNA-dependent RNA polymerase creates an RNA primer, of about 10 bases long, on both the newly separated leading and lagging strands, once for the leading strand and once per Okazaki fragment (about 1000 base pairs long) on the lagging strand. The RNA primer attached to its DNA template is called A-form DNA. (Normal DNA is called B-form DNA.) In prokaryotes primase creates an RNA primer at the beginning of the newly separated leading and lagging strands. DNA polymerase enzymes cannot bind directly to single-stranded DNA and these primers provide a short chain of nucleotides that give the correct configuration to allow the active site of DNA polymerase to fit on and begin elongation.

ElongationThe leading and lagging strands are anti-parallel. In the leading strand nucleotide synthesis (catalysed by DNA polymerase epsilon in eukaryotes and by DNA polymerase III in prokaryotes) proceeds in the 5 to 3 direction (3 to 5 direction on the template strand) and makes a continuous complementary strand. Synthesis of the other strand in the opposite direction cannot occur at the same time so replication of the lagging strand is discontinuous. It involves making short discrete nucleotide chains, called Okazaki fragments, that are then joined by DNA repair enzymes, such as DNA polymerase I and ligase, so it is not made in one continuous strand.This can only happen once a sufficient length of DNA has been unwound so replication of this strand lags behind that of the leading strand.

RNAse H enzymes remove the unstable RNA primers from the newly synthesised fragments and replace them with DNA fragments.DNA ligase (aided by polymerase I in prokaryotes) enzyme connects the Okazaki fragments, closing the gaps between their sugar-phosphate backbones by catalysing the formation of phosphodiester bonds.Proofreading enzymes correct any mistakes due to insertion of incorrect bases.Retrieved from http://www.contentextra.com/lifesciences/files/topicguides/Topic-guide-7.3-DNA-replication.pdf

Mutagenesis and DNA Repair Mechanisms

This rare albino alligator must have the specific "instructions," or DNA, to have this quality. The cause of albinism is a mutation in a gene for melanin, a protein found in skin and eyes. Such a mutation may result in no melanin production at all or a significant decline in the amount of melanin.What causes albinism?

A change in the sequence of bases in DNA or RNA is called a mutation. Does the word mutation make you think of science fiction and bug-eyed monsters? Think again. Everyone has mutations. In fact, most people have dozens or even hundreds of mutations in their DNA. Mutations are essential for evolution to occur. They are the ultimate source of all new genetic materialnew alleles in a species. Although most mutations have no effect on the organisms in which they occur, some mutations are beneficial. Even harmful mutations rarely cause drastic changes in organisms.

Causes of MutationMutations have many possible causes. Some mutations seem to happen spontaneously without any outside influence. They occur when mistakes are made during DNA replication or transcription. Other mutations are caused by environmental factors. Anything in the environment that can cause a mutation is known as a mutagen. Examples of mutagens are pictured in Figure 1.

Figure 1 Examples of Mutagens. Types of mutagens include radiation, chemicals, and infectious agents. Do you know of other examples of each type of mutagen shown here?

Types of MutationsThere are a variety of types of mutations. Two major categories of mutations are germline mutations and somatic mutations. Germline mutations occur in gametes. These mutations are especially significant because they can be transmitted to offspring and every cell in the offspring will have the mutation. Somatic mutations occur in other cells of the body. These mutations may have little effect on the organism because they are confined to just one cell and its daughter cells. Somatic mutations cannot be passed on to offspring. Mutations also differ in the way that the genetic material is changed. Mutations may change the structure of a chromosome or just change a single nucleotide.

What does radiation contamination do? It mutates DNA. The Chernobyl disaster was a nuclear accident that occurred on April 26, 1986. It is considered the worst nuclear power plant accident in history. A Russian publication concludes that 985,000 excess cancers occurred between 1986 and 2004 as a result of radioactive contamination. The 2011 report of the European Committee on Radiation Risk calculates a total of 1.4 million excess cancers occurred as a result of this contamination.

Chromosomal AlterationsChromosomal alterations are mutations that change chromosome structure. They occur when a section of a chromosome breaks off and rejoins incorrectly or does not rejoin at all. Possible ways these mutations can occur are illustrated in Figure 2.

Figure 2 Chromosomal Alterations. Chromosomal alterations are major changes in the genetic material.

Point MutationsMutations A point mutation is a change in a single nucleotide in DNA. This type of mutation is usually less serious than a chromosomal alteration. An example of a point mutation is a mutation that changes the codon UUU to the codon UCU. Point mutations can be silent, missense, or nonsense mutations, as shown in Table 1. The effects of point mutations depend on how they change the genetic code.

Table 1: Point Mutations and Their Effects

Frameshift MutationsA frameshift mutation is a deletion or insertion of one or more nucleotides that changes the reading frame of the base sequence. Deletions remove nucleotides, and insertions add nucleotides. Consider the following sequence of bases in RNA: AUG-AAU-ACG-GCU = start-asparagine-threoninealanine Now, assume an insertion occurs in this sequence. Lets say an A nucleotide is inserted after the start codon AUG: AUG-AAA-UAC-GGC-U = start-lysine-tyrosine-glycineEven though the rest of the sequence is unchanged, this insertion changes the reading frame and thus all of the codons that follow it. As this example shows, a frameshift mutation can dramatically change how the codons in mRNA are read. This can have a drastic effect on the protein product.

Spontaneous MutationsThere are five common types of spontaneous mutations. These are described in the Table 2 below.

Table 7.6: Spontaneous Mutations Described

Effects of MutationsThe majority of mutations have neither negative nor positive effects on the organism in which they occur. These mutations are called neutral mutations. Examples include silent point mutations. They are neutral because they do not change the amino acids in the proteins they encode. Many other mutations have no effect on the organism because they are repaired before protein synthesis occurs. Cells have multiple repair mechanisms to fix mutations in DNA. One way DNA can be repaired is illustrated in Figure 3. If a cells DNA is permanently damaged and cannot be repaired, the cell is likely to be prevented from dividing.

Figure 3: DNA Repair Pathway. This flow chart shows one way that damaged DNA is repaired in E. coli bacteria.

Is this rat hairless?Yes. Why? The result of a mutation, a change in the DNA sequence. The effects of mutations can vary widely, from being beneficial, to having no effect, to having lethal consequences, and every possibility in between.

Beneficial MutationsSome mutations have a positive effect on the organism in which they occur. They are called beneficial mutations. They lead to new versions of proteins that help organisms adapt to changes in their environment. Beneficial mutations are essential for evolution to occur. They increase an organisms changes of surviving or reproducing, so they are likely to become more common over time. There are several well-known examples of beneficial mutations. Here are just two:1. Mutations in many bacteria that allow them to survive in the presence of antibiotic drugs. The mutations lead to antibiotic-resistant strains of bacteria. 2. A unique mutation is found in people in a small town in Italy. The mutation protects them from developing atherosclerosis, which is the dangerous buildup of fatty materials in blood vessels. The individual in which the mutation first appeared has even been identified.

Harmful MutationsImagine making a random change in a complicated machine such as a car engine. The chance that the random change would improve the functioning of the car is very small. The change is far more likely to result in a car that does not run well or perhaps does not run at all. By the same token, any random change in a genes DNA is likely to result in a protein that does not function normally or may not function at all. Such mutations are likely to be harmful. Harmful mutations may cause genetic disorders or cancer.

Cancer is a disease in which cells grow out of control and form abnormal masses of cells. It is generally caused by mutations in genes that regulate the cell cycle. Because of the mutations, cells with damaged DNA are allowed to divide without limits. Cancer genes can be inherited. A genetic disorder is a disease caused by a mutation in one or a few genes. A human example is cystic fibrosis. A mutation in a single gene causes the body to produce thick, sticky mucus that clogs the lungs and blocks ducts in digestive organs.

Genetic DisordersMany genetic disorders are caused by mutations in one or a few genes. Other genetic disorders are caused by abnormal numbers of chromosomes. Genetic Disorders Caused by Mutations Table 3 lists several genetic disorders caused by mutations in just one gene. Some of the disorders are caused by mutations in autosomal genes, others by mutations in X-linked genes. Which disorder would you expect to be more common in males than females?

Table 3: Genetic Disorders Caused by Mutations in One Gene

Few genetic disorders are controlled by dominant alleles. A mutant dominant allele is expressed in every individual who inherits even one copy of it. If it causes a serious disorder, affected people may die young and fail to reproduce. Therefore, the mutant dominant allele is likely to die out of the population.A mutant recessive allele, such as the allele that causes sickle cell anemia (see Figure 7.43), is not expressed in people who inherit just one copy of it. These people are called carriers. They do not have the disorder themselves, but they carry the mutant allele and can pass it to their offspring. Thus, the allele is likely to pass on to the next generation rather than die out.

Figure 5 Sickle-Shaped and Normal Red Blood Cells. Sickle cell anemia is an autosomal recessive disorder. The mutation that causes the disorder affects just one amino acid in a single protein, but it has serious consequences for the affected person. This photo shows the sickle shape of red blood cells in people with sickle cell anemia.

Chromosomal DisordersMistakes may occur during meiosis that result in nondisjunction. This is the failure of replicated chromosomes to separate during meiosis (the animation at the link below shows how this happens). Some of the resulting gametes will be missing a chromosome, while others will have an extra copy of the chromosome. If such gametes are fertilized and form zygotes, they usually do not survive. If they do survive, the individuals are likely to have serious genetic disorders.Table 4 lists several genetic disorders that are caused by abnormal numbers of chromosomes. Figure 7.44 shows a karyotype for trisomy 21 or Downs Syndrome. Most chromosomal disorders involve the X chromosome. Look back at the X and Y chromosomes and you will see why. The X and Y chromosomes are very different in size, so nondisjunction of the sex chromosomes occurs relatively often.

Table 4: Genetic Disorders Caused by Abnormal Number of Chromosomes

Figure 6 Trisomy 21 (Down Syndrome) Karyotype. A karyotype is a picture of a cell's chromosomes. Note the extra chromosome 21. (right) Child with Down syndrome, exhibiting characteristic facial appearance.

Diagnosing Genetic DisordersA genetic disorder that is caused by a mutation can be inherited. Therefore, people with a genetic disorder in their family may be concerned about having children with the disorder. Professionals known as genetic counselors can help them understand the risks of their children being affected. If they decide to have children, they may be advised to have prenatal (before birth) testing to see if the fetus has any genetic abnormalities. One method of prenatal testing is amniocentesis. In this procedure, a few fetal cells are extracted from the fluid surrounding the fetus, and the fetal chromosomes are examined.

Treating Genetic DisordersThe symptoms of genetic disorders can sometimes be treated, but cures for genetic disorders are still in the early stages of development. One potential cure that has already been used with some success is gene therapy. This involves inserting normal genes into cells with mutant genes.

Mutations are caused by environmental factors known as mutagens. Types of mutagens include radiation, chemicals, and infectious agents. Germline mutations occur in gametes. Somatic mutations occur in other body cells. Chromosomal alterations are mutations that change chromosome structure. Point mutations change a single nucleotide. Frameshift mutations are additions or deletions of nucleotides that cause a shift in the reading frame. Mutations are essential for evolution to occur because they increase genetic variation and the potential for individuals to differ. The majority of mutations are neutral in their effects on the organisms in which they occur. Beneficial mutations may become more common through natural selection. Harmful mutations may cause genetic disorders or cancer. Many genetic disorders are caused by mutations in one or a few genes. Other genetic disorders are caused by abnormal numbers of chromosomes. Summary

Retrieved from:www.ck-12.org

http://www.boyertownasd.org/cms/lib07/PA01916192/Centricity/Domain/743/D.%20Chapter%207%20Lesson%204-Mutations.pdf

http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asphttp://www.dnalc.org/resources/3d/17-sicklecell.html

http://www.kqed.org/quest/television/genetic-testing-through-the-web.

http://genetics.wustl.edu/bio5491/files/2016/01/DNA-mutagenesis-lecture-.pdf

http://www.biostudio.com/d_%20Nonsense%20Suppression%20I%20Nonsense%20Mutation.htm

Transcription

To transcribe means "to paraphrase or summarize in writing." The information in DNA is transcribed - or summarized - into a smaller version - RNA - that can be used by the cell. This process is called transcription. Transcription is the first part of the central dogma of molecular biology: DNA RNA. It is the transfer of genetic instructions in DNA to mRNA. During transcription, a strand of mRNA is made that is complementary to a strand of DNA. Figure 1 shows how this occurs.

Figure 1 Overview of Transcription. Transcription uses the sequence of bases in a strand of DNA to make a complementary strand of mRNA. Triplets are groups of three successive nucleotide bases in DNA. Codons are complementary groups of bases in mRNA.

Steps of TranscriptionTranscription takes place in three steps: initiation, elongation, and termination. The steps are illustrated in Figure 2.1. Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can read the bases in one of the DNA strands. The enzyme is ready to make a strand of mRNA with a complementary sequence of bases.2. Elongation is the addition of nucleotides to the mRNA strand. 3. Termination is the ending of transcription, and occurs when RNA polymerase crosses a stop (termination) sequence in the gene. The mRNA strand is complete, and it detaches from DNA.

Figure 2. Steps of Transcription. Transcription occurs in the three steps - initiation, elongation, and termination - shown here.

Source:www.ck-12.org

Processing RNA

In prokaryotes, no RNA processing is necessary: the nascent RNA is usually the mRNA. In eukaryotes, the nascent RNA is called primary transcript-RNA needs to be processed and transported to the cytoplasm for translation to occur.

Fig. 1 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes

Splicing removes introns from mRNA (see Figure 2). Introns are regions that do not code for proteins. The remaining mRNA consists only of regions that do code for proteins, which are called exons. Ribonucleoproteins are nucleoproteins that contain RNA. Small nuclear ribonuclearproteins are involved in pre-mRNA splicing.

Figure 2 Splicing. Splicing removes introns from mRNA. UTR is an untranslated region of the mRNA.

Editing changes some of the nucleotides in mRNA. For example, the human protein called APOB, which helps transport lipids in the blood, has two different forms because of editing. One form is smaller than the other because editing adds a premature stop signal in mRNA. Polyadenylation adds a tail to the mRNA. The tail consists of a string of As (adenine bases). It signals the end of mRNA. It is also involved in exporting mRNA from the nucleus. In addition, the tail protects mRNA from enzymes that might break it down.

Figure 3 The ends of eukaryotic mRNAs

Questions

Why is the mRNA not equal in length to the DNA it was transcribed from? 1) the mRNA was longer because it has a Poly A tail 2) The mRNA was longer because it contains only introns 3) The DNA was shorter because it does not have the Methylated cap 4) The mRNA was shorter because of Intron splicing Which nucleotides signal the 5 end of an intron splice site? 1. AT 2. GU 3. AG 4. GG

Retrieved from:

http://vcell.ndsu.edu/animations/mrnasplicing/movie-flash.htm.

http://www.boyertownasd.org/cms/lib07/PA01916192/Centricity/Domain/743/A.%20Chapter%207%20Lesson%201-From%20DNA%20to%20Proteins.pdf

http://www.csun.edu/~cmalone/pdf360/Ch13-2RNAprocess.pdf

TranslationTranslation is the second part of the central dogma of molecular biology: RNA Protein. It is the process in which the genetic code in mRNA is read to make a protein. Figure 1 shows how this happens. After mRNA leaves the nucleus, it moves to a ribosome, which consists of rRNA and proteins. The ribosome reads the sequence of codons in mRNA. Molecules of tRNA bring amino acids to the ribosome in the correct sequence.

To understand the role of tRNA, you need to know more about its structure. Each tRNA molecule has an anticodon for the amino acid it carries. An anticodon is complementary to the codon for an amino acid. For example, the amino acid lysine has the codon AAG, so the anticodon is UUC. Therefore, lysine would be carried by a tRNA molecule with the anticodon UUC. Wherever the codon AAG appears in mRNA, a UUC anticodon of tRNA temporarily binds. While bound to mRNA, tRNA gives up its amino acid. Bonds form between the amino acids as they are brought one by one to the ribosome, forming a polypeptide chain. The chain of amino acids keeps growing until a stop codon is reached. To see how this happens, go the link below.

Figure 1: Translation. Translation of the codons in mRNA to a chain of amino acids occurs at a ribosome. Find the different types of RNA in the diagram. What are their roles in translation?

After a polypeptide chain is synthesized, it may undergo additional processes. For example, it may assume a folded shape due to interactions among its amino acids. It may also bind with other polypeptides or with different types of molecules, such as lipids or carbohydrates. Many proteins travel to the Golgi apparatus to be modified for the specific job they will do. Source:www.ck-12.org

GENE REGULATION

Gene regulation is a label for the cellular processes that control the rate and manner of gene expression. A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced. Often, one gene regulator controls another, and so on, in agene regulatory network. Gene regulation is essential forviruses,prokaryotesandeukaryotesas it increases the versatility and adaptability of anorganismby allowing the cell to express protein when needed.

WHAT IS GENE REGULATION?

Although as early as 1951,Barbara McClintockshowed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color formation of maize seeds, the first discovery of a gene regulation system is widely considered to be the identification in 1961 of thelacoperon, discovered byJacques Monod, in which some enzymes involved inlactosemetabolism are expressed byE. colionly in the presence of lactose and absence of glucose.

Any step of gene expression may be modulated, from the DNA-RNAtranscriptionstep topost-translational modificationof a protein. The following is a list of stages where gene expression is regulated, the most extensively utilized point is Transcription Initiation:Chromatin domainsTranscriptionPost-transcriptional modificationRNA transportTranslationmRNA degradation

WHY IS GENE EXPRESSION REGULATED? Genes can't control an organism on their own; rather, they mustinteract with and respond to the organism's environment. Some genes are constitutive, or always "on," regardless of environmental conditions. Such genes are among the most important elements of a cell's genome, and they control the ability of DNA to replicate, express itself, and repair itself. These genes also control protein synthesis and much of an organism's central metabolism. It turns out that the regulation of such genes differs between PROKARYOTES and EUKARYOTES. For prokaryotes, mostregulatory proteins are negative and therefore turn genesoff and in eukaryotes, the default state of gene expression is off rather than on.

Prokaryotic gene regulationFor prokaryotes, mostregulatory proteins are negative and therefore turn genesoff. Here, the cells rely on proteinsmall molecule binding, in which a ligand or small molecule signals the state of the cell and whether gene expression is needed. Therepressororactivator proteinbinds near its regulatory target: the gene. Some regulatory proteins must have a ligand attached to them to be able to bind, whereas others are unable to bind when attached to a ligand. In prokaryotes, most regulatory proteins are specific to one gene, although there are a few proteins that act more widely. For instance, some repressors bind near the start of mRNA production for anentire operon, or cluster of coregulated genes.

Furthermore, some repressors have a fine-tuning system known as attenuation, which uses mRNA structure to stop both transcription and translation depending on the concentration of an operon's end-product enzymes. (In eukaryotes, there is no exact equivalent ofattenuation, because transcription occurs in the nucleus and translation occurs in the cytoplasm, making this sort of coordinated effect impossible.) Yet another layer of prokaryotic regulation affects the structure ofRNA polymerase, which turns on large groups of genes. Here, the sigma factor of RNA polymerase changes several times to produce heat- and desiccation-resistant spores. Here, the articles on prokaryotic regulation delve into each of these topics, leading to primary literature in many cases.

PROKARYOTIC GENE REGULATION (TRANSCRIPTION REGULATION) DIAGRAM

Eukaryotic gene regulation Unlike prokaryotes, multiple gene-regulating mechanisms operate in the nucleus before and after RNA transcription, and in the cytoplasm both before and after translation.Histonesare small proteins packed inside the molecular structure of the DNA double helix. Tight histone packing prevents RNA polymerase from contacting and transcribing the DNA. This type of overall control of protein synthesis is regulated by genes that control the packing density of histones.X-chromosome inactivationoccurs when dense packing of the X chromosome in females totally prevents its function even in interphase. This type of inactivation is inherited and begins during embryonic development, where one of the X chromosomes is randomly packed, making it inactive for life.

Activator-enhancer complexis unique in eukaryotes because they normally have to be activated to begin protein synthesis, which requires the use oftranscription factorsand RNA polymerase. In general, the process of eukaryotic protein synthesis involves four steps:1. Activators, a special type of transcription factor, bind toenhancers, which are discrete DNA units located at varying points along the chromosome.2. The activator-enhancer complex bends the DNA molecule so that additional transcription factors have better access to bonding sites on the operator.3. The bonding of additional transcription factors to the operator allows greater access by the RNA polymerase, which then begins the process of transcription.4. Silencersare a type of repressor protein that blocks transcription at this point by bonding with particular DNA nucleotide sequences.

Eukaryotic gene expression involves many steps, and almost all of them can be regulated. Different genes are regulated at different points, and its not uncommon for a gene (particularly an important or powerful one) to be regulated at multiple steps.Chromatin accessibility. The structure of chromatin (DNA and its organizing proteins) can be regulated. More open or relaxed chromatin makes a gene more available for transcription.Transcription. Transcription is a key regulatory point for many genes. Sets oftranscription factorproteins bind to specific DNA sequences in or near a gene and promote or repress its transcription into an RNA.RNA processing. Splicing, capping, and addition of a poly-A tail to an RNA molecule can be regulated, and so can exit from the nucleus. Different mRNAs may be made from the same pre-mRNA byalternative splicing.

RNA stability. The lifetime of an mRNA molecule in the cytosol affects how many proteins can be made from it. Small regulatory RNAs calledmiRNAscan bind to target mRNAs and cause them to be chopped up.Translation. Translation of an mRNA may be increased or inhibited by regulators. For instance, miRNAs sometimes block translation of their target mRNAs (rather than causing them to be chopped up).Protein activity. Proteins can undergo a variety of modifications, such as being chopped up or tagged with chemical groups. These modifications can be regulated and may affect the activity or behavior of the protein.

The processing and packaging of RNA both in the nucleus and cytoplasm provides two more opportunities for gene regulation to occur after transcription but before translation.Adding extra nucleotides as a protective cap and tail to the RNA identifies the RNA as an mRNA by the ribosomes, and prevents degradation by cell enzymes as it moves from the nucleus into the cytoplasm.RNA splicingoccurs when gaps of nonprotein-code-carrying nucleotides calledinteronsare removed from the code-carrying nucleotides, calledexons, which are then connected to shorten the RNA molecule for conversion into tRNA and rRNA. The number of interons regulates the speed at which the RNA can be processed.After the extra nucleotides have been added as a cap and tail and the RNA has been spliced, it moves to the cytoplasm where additional mechanisms of gene regulation exist.

The longevity of the individual mRNA molecule determines how many times it can be used and reused to create proteins. In eukaryotes, the mRNA tends to be stable, which means it can be used multiple times; which is efficient, but it prevents eukaryotes from making rapid response changes to environmental disruptions. The mRNA of prokaryotes is unstable, allowing for the creation of new mRNA, which has more opportunities to adjust for changing environmental conditions.Inhibitory proteinsprevent the translation of mRNA. They are made inactive when bonded with the substance for which they are trying to block production.Post-translationcontrol involves the selective cutting and breakdown of proteins that prevent the formation of the final product. In both cases, the hormone or enzyme required to finish or activate the final product may be rendered inactive.

EXAMPLES OF GENE REGULATION Enzyme inductionis a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme.TheLac operonis an interesting example of how gene expression can be regulated. Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV).GAL4 is a transcriptional activator that controls the expression of GAL1, GAL7, and GAL10 (all of which code for the metabolic of galactose in yeast). TheGAL4/UAS systemhas been used in a variety of organisms across various phyla to study gene expression

ENZYME INDUCTION Enzymeinductionis a process in which a molecule (e.g. a drug) induces (i.e. initiates or enhances) the expression of an enzyme. An enzyme inducer is a type of drug which binds to an enzyme and increases its metabolic activity. Many of the enzymes involved in drug metabolism may be up-regulated by exposure to drugs and environmental chemicals leading to increased rates of metabolism. This phenomenon is known asenzymeinduction.Enzymeinductionis a process where production of an enzyme is triggered or increased in response to changes in the environment that surrounds an individual You do not have access to view this node. The increase in enzyme expression creates a chain reaction as the enzyme begins to act in the body.

ENZYME INDUCTION DIAGRAM

WHAT IS ENZYME REPRESSION? Inductionand repression are linked in that they both focus on the binding of a molecule known as RNA polymerase to DNA. Particularly, the RNA polymerase binds to a region that is immediately "upstream" from the region of DNA that code for a protein. The binding region is termed the operator. The operator acts to position the polymerase correctly, so that the molecule can then begin to move along the DNA, interpreting the genetic information as it moves along. The three-dimensional shape of the operator region manipulates the binding of the RNA polymerase. The configuration of the operator can be changed by the presence of molecules called Effectors.

What is the process of enzyme induction? Enzymeinductionis a process where an enzyme is contrived in response to the presence of a specific molecule. This molecule is termed an inducer. Basically, an inducer molecule is a compound that the enzyme acts upon. In theinductionprocess, the inducer molecule merges with another molecule, which is called the repressor (a chemical compound that is designed to limit or prevent enzyme production, so there are no obstacles to enzyme production). The binding of the inducer to the repressor obstructs the function of the repressor, which is to bind to a specific region called an operator. The operator is the site to which another molecule, known as ribonucleic acid (RNA) polymerase, binds and begins the transcription (transfer of genetic information from DNA to RNA) of the gene to produce the so-called messenger RNA that performs as a guide for the subsequent production of protein.

Thus, the binding of the inducer to the repressor keeps the repressor from averting transcription, and so the gene coding for the inducible enzyme is transcribed. Repression of transcription is basically the default behavior, which is dominated once the inducing molecule is present. In bacteria, the lactose (lac) operon is a very well characterized system that operates on the basis ofinduction. An operon is a single unit of physically adjacent genes that function together under the control of a single operator gene.

LAC OPERON Thelac operon(lactose operon) is anoperonrequired for the transport and metabolism oflactosein Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, thelac operon allows for the effective digestion oflactosewhen glucose is not available. Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as a sugar source for the bacterium, the three genes of the lac operon can be expressed and their subsequent proteins translated:lacZ,lacY, andlacA. The gene product oflacZis-galactosidasewhich cleaves lactose, a disaccharide, intoglucoseandgalactose.lacYencodesBeta-galactoside permease, a protein which becomes embedded in the cytoplasmic membrane to enable transport of lactose into the cell. Finally,lacAencodes-galactoside transacetylase.

It would be wasteful to produce the enzymes when there is no lactose available or if there is a more preferable energy source available, such as glucose. Thelacoperon uses a two-part control mechanism to ensure that the cell expends energy producing the enzymes encoded by thelacoperon only when necessary. In the absence of lactose, thelacrepressorhalts production of the enzymes encoded by thelacoperon. In the presence of glucose, thecatabolite activator protein(CAP), required for production of the enzymes, remains inactive, andEIIAGlcshuts down lactose permease to prevent transport of lactose into the cell. This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known asdiauxie.

STRUCTURE OF LAC OPERON Thelacoperon contains three genes:lacZ,lacY, andlacA. These genes are transcribed as a single mRNA, under control of one promoter.Genes in thelacoperon specify proteins that help the cell utilize lactose.lacZencodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis. Similarly,lacYencodes a membrane-embedded transporter that helps bring lactose into the cell.In addition to the three genes, thelacoperon also contains a number of regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind, controlling transcription of the operon.

Thepromoteris the binding site for RNA polymerase, the enzyme that performs transcription.Theoperatoris a negative regulatory site bound by thelacrepressor protein. The operator overlaps with the promoter, and when thelacrepressor is bound, RNA polymerase cannot bind to the promoter and start transcription.TheCAP binding siteis a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.

STRUCTURE OF LAC OPERON

The lac repressor

Thelacrepressor is a protein that represses (inhibits) transcription of thelacoperon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, thelacrepressor gets in RNA polymerase's way and keeps it from transcribing the operon.When lactose is not available, thelacrepressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, thelacrepressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon.

This change in thelacrepressor is caused by the small moleculeallolactose, an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to thelacrepressor and makes it change shape so it can no longer bind DNA.Allolactose is an example of aninducer, a small molecule that triggers expression of a gene or operon. Thelacoperon is considered aninducible operonbecause it is usually turned off (repressed), but can be turned on in the presence of the inducer allolactose.

Catabolite activator protein (cap) When lactose is present, thelacrepressor loses its DNA-binding ability. This clears the way for RNA polymerase to bind to the promoter and transcribe thelacoperon. As it turns out, RNA polymerase alone does not bind very well to thelacoperon promoter. It might make a few transcripts, but it won't do much more unless it gets extra help fromcatabolite activator protein(CAP). CAP binds to a region of DNA just before thelacoperon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription.

CAP isn't always active (able to bind DNA). Instead, it's regulated by a small molecule calledcyclic AMP(cAMP). cAMP is a "hunger signal" made byE. coliwhen glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive.CAP is only active when glucose levels are low (cAMP levels are high). Thus, thelacoperon can only be transcribed at high levels when glucose is absent. This strategy ensures that bacteria only turn on thelacoperon and start using lactose after they have used up all of the preferred energy source (glucose).

Glucose present, lactose absent: No transcription of thelacoperon occurs. That's because thelacrepressor remains bound to the operator and prevents transcription by RNA polymerase. Also, cAMP levels are low because glucose levels are high, so CAP is inactive and cannot bind DNA.

Glucose present, lactose present: Low-level transcription of thelacoperon occurs. Thelacrepressor is released from the operator because the inducer (allolactose) is present. cAMP levels, however, are low because glucose is present. Thus, CAP remains inactive and cannot bind to DNA, so transcription only occurs at a low, leaky level.

Glucose absent, lactose absent: No transcription of thelacoperon occurs. cAMP levels are high because glucose levels are low, so CAP is active and will be bound to the DNA. However, thelacrepressor will also be bound to the operator (due to the absence of allolactose), acting as a roadblock to RNA polymerase and preventing transcription.

Glucose absent, lactose present: Strong transcription of thelacoperon occurs. Thelacrepressor is released from the operator because the inducer (allolactose) is present. cAMP levels are high because glucose is absent, so CAP is active and bound to the DNA. CAP helps RNA polymerase bind to the promoter, permitting high levels of transcription.

VIRAL REPLICATION Replication of viruses primarily involves the multiplication of the viral genome. Replication also involves synthesis of viralmessenger RNA(mRNA) from "early"genes(with exceptions for positive sense RNA viruses), viralproteinsynthesis, possible assembly of viral proteins, then viral genome replication mediated by early or regulatory proteinexpression. This may be followed, for complex viruses with larger genomes, by one or more further rounds of mRNA synthesis: "late" gene expression is, in general, necessary for structural orvirionproteins. Viral replication usually takes place in thecytoplasm.

SCHEMATIC SHOWING ANTISENSE DNA STRANDS CAN INTERFERE WITH PROTEIN TRANSLATION

Viruses that replicate via RNA intermediates need anRNA-dependent RNA-polymeraseto replicate their RNA, but animal cells do not seem to possess a suitableenzyme. Therefore, this type of animal RNAvirusneeds to code for an RNA-dependentRNA polymerase. No viral proteins can be made until viral messenger RNA is available; thus, the nature of the RNA in the virion affects the strategy of the virus: In plus-stranded RNA viruses, the virion (genomic) RNA is the same sense as mRNA and so functions as mRNA. This mRNA can be translated immediately uponinfectionof thehostcell . Examples: poliovirus (picornavirus), togaviruses, and flaviviruses.

Viral gene expression regulation refers to any of the processes by which cytoplasmic factors influence the differential control of gene action in viruses. The interplay of the viral genome with the host metabolic machinery involves modifications in both gene expression and regulation. Retroviruses have adapted themselves to use this machinery while maintaining the cell integrity, which is essential to preserve their survival. Consequently, there can be variable host pathogenicity associated with several diseases such as malignancies, immunodeficiencies, and neurological disorders. This book describes current research in the field, and gives a better understanding of the retroviral gene expression regulation that is essential to develop prevention and therapeutic strategies in the future.

GAL4 SYSTEM The GAL4-UAS systemis abiochemicalmethod used to studygene expressionand function in organisms such as thefruit fly. It has also been adapted to studyreceptorchemical-binding functionsin vitroincell culture. It was developed byAndrea BrandandNorbert Perrimonin 1993and is considered a powerful technique for studying theexpression of genes.The system has two parts: the GAL4 gene, encoding theyeasttranscription activatorproteinGAL4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate genetranscription.

The GAL4 system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge. Geneticists have created genetic varieties of model organisms (typically fruit flies), calledGAL4 lines, each of which expresses GAL4 in some subset of the animal's tissues. For example, some lines might express GAL4 only in muscle cells, or only in nerves, or only in the antennae, and so on. For fruit flies in particular, there are tens of thousands of such lines, with the most useful expressing GAL4 in only a very specific subset of the animalperhaps, for example, only thoseneuronsthat connect two specific compartments of the fly's brain. The presence of GAL4, by itself, in these cells has little or no effect, since GAL4's main effect is to bind to a UAS region, and most cells have no (or innocuous) UAS regions.

Since GAL4 by itself is not visible, and has little effect on cells, the other necessary part of this system are the "reporter lines". These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells thatareproducing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. This may report to the investigator which cells are expressing GAL4, hence the term "reporter line", but genes intended to manipulate the cell behavior are often used as well.

Typicalreporter genesinclude:Fluorescent proteins likegreen(GFP) or red fluorescent proteins (RFP), which allow scientists to see which cells express GAL4Channelrhodopsin, which allows light-sensitive triggering of nerve cellsHalorhodopsin, which conversely allows light to suppress the firing of neuronsShibire, which shuts neurons off, but only at higher temperatures (30C and above). Flies with this gene can be raised and tested at lower temperatures where their neurons will behave normally. Then the body temperature of the flies can be raised (since they arecold-blooded), and these neurons turn off.[3]If the fly's behavior changes, this gives a strong clue to what those neurons do.GECI (GeneticallyEncodedCalciumIndicator), often a member of theGCaMPfamily of proteins. These proteins glow when exposed tocalcium, which, in most cells, happens when the neuron fires. This allows scientists to take pictures, or movies, that show the nervous system in operation.

RNA PROCESSING

RNA serves a multitude of functions within cells. These functions are primarily involved in converting the genetic information contained in a cell's DNA into the proteins that determine the cell's structure and function. All RNAs are originally transcribed from DNA by RNA polymerases, which are specializedenzymecomplexes, but most RNAs must be further modified or processed before they can carry out their roles. Thus, RNA processing refers to any modification made to RNA between its transcription and its final function in the cell. These processing steps include the removal of extra sections of RNA, specific modifications of RNA bases, and modifications of the ends of the RNA.

Types of rna There are different types of RNA, each of which plays a specific role, including specifying the amino acid sequence of proteins (performed by messenger RNAs, or mRNAs), organizing and catalyzing the synthesis of proteins (ribosomal RNAs or rRNAs), translating codons in the mRNA into amino acids (transfer RNAs or tRNAs) and directing many of the RNA processing steps (performed by small RNAs in the nucleus, called snRNAs and snoRNAs).

All of these types of RNAs begin as primary transcripts copied from DNA by one of the RNA polymerases. One of the features that separateseukaryotesandprokaryotesis that eukaryotes isolate their DNA inside a nucleus while protein synthesis takes place in the cytoplasm. This separates the processes of transcription and translation in space and time. Prokaryotes, which lack a nucleus, can translate an mRNA as soon as it is transcribed by RNA polymerase. As a consequence, there is very little processing of prokaryotic mRNAs. By contrast, in eukaryotic cells many processing steps occur between mRNA transcription and translation. Unlike the case of mRNAs, both eukaryotes and prokaryotes process their rRNAs and tRNAs in broadly similar ways.

TYPES OF RNA PROCESSING There are three main types of RNA processing events: trimming one or both of the ends of the primary transcript to the mature RNA length; removing internal RNA sequences by a process called RNA splicing; and modifying RNA nucleotides either at the ends of an RNA or within the body of the RNA. We will briefly examine each of these and then discuss how they are applied to the various types of cellular RNAs.Almost all RNAs have extra sequences at one or both ends of the primary transcripts that must be removed. The removal of individual nucleotides from the ends of the RNA strand is carried out by any of several ribonucleases (enzymes that cut RNA), called exoribonucleases. An entire section of RNA sequence can be removed by cleavage in the middle of an RNA strand. The enzymes responsible for the cleavage in this location are called endoribonucleases. Each of these ribonucleases is targeted so that it only cleaves particular RNAs at particular places.RNA splicing is similar to trimming in that it removes extra RNA sequences, but it is different because the sequence is removed from the middle of an RNA and the two flanking pieces are joined together again (see figure). The part of the RNA that is removed is called an intron, whereas the two pieces that are joined together, or spliced, are called exons. Just as with the cleavage enzymes, the splicing machinery recognizes particular sites within the RNA, in this case the junctions between exons and introns, and cleaves and rejoins the RNA at those positions.Modification of RNA nucleotides can occur at the ends of an RNA molecule or at internal positions. Modification of the ends can protect the RNA from degradation by exoribonucleases and can also act as a signal to guide the transport of the molecule to a particular subcellular compartment. Some internal modifications, particularly of tRNAs and rRNAs, are necessary for these RNAs to carry out their functions in protein synthesis. Some internal modifications of mRNAs change the sequence of the message and so change the amino acid sequence of the protein coded for by the mRNA. This process is called RNA editing. As with the other types of RNA processing, the enzymes that modify RNAs are directed to specific sites on the RNA.

Processing of Various Classes of RNAs

Ribosomal RNAs are synthesized as long primary transcripts that contain several different rRNAs separated by spacer regions .The individual rRNAs are cut apart by endoribonucleases that cleave within the spacer regions. Other enzymes then trim the ends to their final length. Ribosomal RNAs are also modified at many specific sites within the RNA. Ribosomal RNA synthesis and processing occurs in a special structure within the nucleus called thenucleolus. The mature rRNAs bind to ribosomal proteins within the nucleolus and the assembled ribosomes are then transported to thecytoplasmto carry out protein synthesis.Transfer RNAs are transcribed individually from tRNA genes. The primary transcripts are trimmed at both the 5and 3("five prime," or "upstream" and "three prime," or "downstream") ends, and several modifications are made to internal bases. Many eukaryotic tRNAs also contain an intron, which must be removed by RNA splicing. The finished tRNAs are then transported from the nucleus to the cytoplasm.Messenger RNAs are transcribed individually from their genes as very long primary transcripts. This is because most eukaryotic genes are divided into many exons separated by introns. Genes may contain from zero to more than sixty introns, with a typical gene having around ten. Introns are spliced out of primary RNA transcripts by a large structure called thespliceosome. The spliceosome does not move along the RNA but is assembled around each intron where it cuts and joins the RNA to remove the intron and connect the exons. This must be done many times on a typical primary transcript to produce the mature mRNA.

In addition to removal of the introns, the mRNA is modified at the 5end by the addition of a special "cap" structure that is later recognized by the translation machinery. The mRNA is also trimmed at the 3end and several hundred adenosine nucleotides are added. This modification, which is called either polyadenylation or poly (A) addition, helps stabilize the 3end against degradation and is also recognized by the translation machinery. Finally, the processed mature mRNA is transported from the nucleus to the cytoplasm.Some RNAs, called small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), are processed in the nucleus and are themselves part of the RNA processing systems in the nucleus. Most snRNAs are involved in mRNA splicing, while most snoRNAs are involved in rRNA cleavage and modification.

RNA Processing and the Human Genome

The fact that most human genes are composed of many exons has some important consequences for the expression of genetic information. First, we now know that many genes are spliced in more than one way, a phenomenon known as alternative splicing. For example, some types of cells might leave out an exon from the final mRNA that is left in by other types of cells, giving it a slightly different function. This means that a single gene can code for more than one protein. Some complicated genes appear to be spliced to give hundreds of alternative forms. Alternative splicing, therefore, can increase the coding capacity of the genome without increasing the number of genes.A second consequence of the exon/intron gene structure is that many human gene mutations affect the splicing pattern of that gene. For example, a mutation in the sequence at an intron/exon junction that is recognized by the spliceosome can cause the junction to be ignored. This causes splicing to occur to the next exon in line, leaving out the exon next to the mutation. This is called exon skipping and it usually results in an mRNA that codes for a nonfunctional protein. Exon skipping and other errors in splicing are seen in many human genetic diseases.

POST-TRANSCRIPTIONAL MODIFICATION Post-transcriptional modificationorCo-transcriptional modificationis the process ineukaryotic cellswhereprimary transcriptRNA is converted intomature RNA. A notable example is the conversion ofprecursor messenger RNAintomaturemessenger RNA(mRNA) that occurs prior to protein translation. The process includes three major steps: addition of a5' cap, addition of a 3' poly-adenylationtail, andsplicing. This process is vital for the correcttranslationof thegenomesofeukaryotesbecause the initial precursor mRNA produced duringtranscriptioncontains bothexons(coding or important sequences involved in translation), andintrons(non-coding sequences)

Example of a signal that directs post-transcriptional processing: the conserved eukaryoticpolyadenylationsignal directs cleavage at the cleavage signal and addition of a poly-A tail to the mRNA transcript

5 processing Capping Capping of the pre-mRNA involves the addition of7-methylguanosine(m7G) to the 5' end. To achieve this, the terminal 5' phosphate requires removal, which is done with the aid of aphosphataseenzyme. The enzymeguanosyl transferasethen catalyses the reaction, which produces thediphosphate5' end. The diphosphate 5' end then attacks the alpha phosphorus atom of aGTPmolecule in order to add theguanineresidue in a 5'5' triphosphate link. The enzyme (guanine-N7-)-methyltransferase ("cap MTase") transfers a methyl group fromS-adenosyl methionineto the guanine ring.[3]This type of cap, with just the (m7G) in position is called a cap 0 structure. Theriboseof the adjacentnucleotidemay also be methylated to give a cap 1. Methylation of nucleotides downstream of the RNA molecule produce cap 2, cap 3 structures and so on. In these cases the methyl groups are added to the 2' OH groups of the ribose sugar. The cap protects the 5' end of the primary RNA transcript from attack byribonucleasesthat have specificity to the 3'5'phosphodiester bonds.

3 processingCleavage and polyadenylation The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 250adenineresidues to form apoly(A) tail. The cleavage and adenylation reactions occur if apolyadenylation signal sequence(5'- AAUAAA-3') is located near the 3' end of the pre-mRNA molecule, which is followed by another sequence, which is usually(5'-CA-3')and is the site of cleavage. AGU-rich sequenceis also usually present further downstream on the pre-mRNA molecule. After the synthesis of the sequence elements, two multisubunitproteinscalledcleavage and polyadenylation specificity factor(CPSF) andcleavage stimulation factor(CStF) are transferred fromRNA Polymerase IIto the RNA molecule. The two factors bind to the sequence elements. A protein complex forms that contains additional cleavage factors and the enzymePolyadenylate Polymerase(PAP). This complex cleaves the RNA between the polyadenylation sequence and the GU-rich sequence at the cleavage site marked by the (5'-CA-3') sequences. Poly(A) polymerase then adds about 200 adenine units to the new 3' end of the RNA molecule usingATPas a precursor. As the poly(A) tail is synthesised, it binds multiple copies of poly(A) binding protein, which protects the 3'end from ribonuclease digestion

Splicing Splicing RNA splicing is the process by whichintrons, regions of RNA that do not code for protein, are removed from the pre-mRNA and the remainingexonsconnected to re-form a single continuous molecule. Exons are sections of mRNA which become "expressed" or translated into a protein. They are the coding portions of a mRNA molecule.[5]Although most RNA splicing occurs after the complete synthesis and end-capping of the pre-mRNA, transcripts with many exons can be spliced co-transcriptionally.[6]The splicing reaction is catalyzed by a large protein complex called thespliceosomeassembled from proteins andsmall nuclear RNAmolecules that recognizesplice sitesin the pre-mRNA sequence. Many pre-mRNAs, including those encodingantibodies, can be spliced in multiple ways to produce different mature mRNAs that encode differentprotein sequences. This process is known asalternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.

TRANSLATION

Translationis the process in whichribosomesin acell'scytoplasmcreateproteins, followingtranscriptionofDNAtoRNAin the cell'snucleus. The entire process is a part ofgene expression.In translation,messenger RNA (mRNA)is decoded by a ribosome, outside the nucleus, to produce a specificamino acidchain, orpolypeptide. The polypeptide laterfoldsinto anactiveprotein and performs its functions in thecell.Theribosomefacilitates decoding by inducing the binding ofcomplementarytRNAanticodonsequences to mRNAcodons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome.Translation proceeds in three phases:Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at thestart codon.Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon. The ribosome then moves (translocates)to the next mRNA codon to continue the process, creating an amino acid chain.Termination: When a stop codon is reached, the ribosome releases the polypeptide.In bacteria, translation occurs in the cytoplasm, where the large and small subunits of theribosomebind to the mRNA. In eukaryotes, translation occurs in thecytosolor across the membrane of theendoplasmic reticulumin a process calledvectorial synthesis. In many instances, the entire ribosome/mRNA complex binds to the outer membrane of therough endoplasmic reticulum(ER); the newly created polypeptide is stored inside the ER for latervesicletransport andsecretionoutside of the cell.

The genetic code

During translation, a cell reads the information in a messenger RNA (mRNA) and uses it to build a protein. Actually, to be a little more techical, an mRNA doesnt always encodeprovide instructions fora whole protein. Instead, what we can confidently say is that it always encodes apolypeptide, or chain of amino acids. In an mRNA, the instructions for building a polypeptide are RNA nucleotides (As, Us, Cs, and Gs) read in groups of three. These groups of three are calledcodons.There are616161codons for amino acids, and each of them is "read" to specify a certain amino acid out of the202020commonly found in proteins. One codon, AUG, specifies the amino acid methionine and also acts as astart codonto signal the start of protein construction.There are three more codons that donotspecify amino acids. Thesestop codons, UAA, UAG, and UGA, tell the cell when a polypeptide is complete. All together, this collection of codon-amino acid relationships is called thegenetic code, because it lets cells decode an mRNA into a chain of amino acids.

Transfer RNAs (tRNAs)Transfer RNAs, ortRNAs, are molecular "bridges" that connect mRNA codons to the amino amino acids they encode. One end of each tRNA has a sequence of three nucleotides called ananticodon, which can bind to specific mRNA codons. The other end of the tRNA carries the amino acid specified by the codons.There are many different types of tRNAs. Each type reads one or a few codons and brings the right amino acid matching those codons.

ribosomal RNA Ribosomesare the structures where polypeptides (proteins) are built. They are made up of protein and RNA (ribosomal RNA, orrRNA). Each ribosome has two subunits, a large one and a small one, which come together around an mRNAkind of like the two halves of a hamburger bun coming together around the patty.The ribosome provides a set of handy slots where tRNAs can find their matching codons on the mRNA template and deliver their amino acids. These slots are called the A, P, and E sites. Not only that, but the ribosome also acts as an enzyme, catalyzing the chemical reaction that links amino acids together to make a chain.

GETTING STARTED: Initiation

Ininitiation, the ribosome assembles around the mRNA to be read and the first tRNA (carrying the amino acid methionine, which matches the start codon, AUG). This setup, called the initiation complex, is needed in order for translation to get started.

Extending the chain: Elongation

Elongationis the stage where the amino acid chain getslonger. In elongation, the mRNA is read one codon at a time, and the amino acid matching each codon is added to a growing protein chain.Each time a new codon is exposed:A matching tRNA binds to the codonThe existing amino acid chain (polypeptide) is linked onto the amino acid of the tRNA via a chemical reactionThe mRNA is shifted one codon over in the ribosome, exposing a new codon for reading

During elongation, tRNAs move through the A, P, and E sites of the ribosome, as shown above. This process repeats many times as new codons are read and new amino acids are added to the chain.

Finishing up: Termination

Terminationis the stage in which the finished polypeptide chain is released. It begins when a stop codon (UAG, UAA, or UGA) enters the ribosome, triggering a series of events that separate the chain from its tRNA and allow it to drift out of the ribosome.After termination, the polypeptide may still need to fold into the right 3D shape, undergo processing (such as the removal of amino acids), get shipped to theright place in the cell, or combine with other polypeptides before it can do its job as a functional protein.

EPIGENETICS

Epigenetics is the study of potentially heritable changes in gene expression (active versus inactive genes) that does not involve changes to the underlying DNA sequence a change inphenotype without a change ingenotype which in turn affects how cells read the genes. Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state. Epigenetic modifications can manifest as commonly as the manner in which cells terminally differentiate to end up as skin cells, liver cells, brain cells, etc. Or, epigenetic change can have more damaging effects that can result in diseases like cancer. At least three systems including DNAmethylation, histone modification andnon-coding RNA(ncRNA)-associated gene silencing are currently considered to initiate and sustain epigenetic change.1New and ongoing research is continuously uncovering the role ofepigeneticsin a variety of human disorders and fatal diseases.

Epigenetic mechanism

One example of an epigenetic change ineukaryoticbiology is the process ofcellular differentiation. Duringmorphogenesis,totipotentstem cellsbecome the variouspluripotentcell linesof theembryo, which in turn become fully differentiated cells. In other words, as a single fertilized egg cell thezygote continues todivide, the resulting daughter cells change into all the different cell types in an organism, includingneurons,muscle cells,epithelium,endotheliumofblood vessels, etc., by activating some genes while inhibiting the expression of others.Historically, some phenomena not necessarily heritable have also been described as epigenetic. For example, epigenetic has been used to describe any modification of chromosomal regions, especiallyhistone modifications, whether or not these changes are heritable or associated with a phenotype. The consensus definition now requires a trait to be heritable for it to be considered epigenetic.

History What began as broad research focused on combining genetics and developmental biology by well-respected scientists including Conrad H. Waddington and Ernst Hadorn during the mid-twentieth century has evolved into the field we currently refer to asepigenetics. The term epigenetics, which was coined by Waddington in 1942, was derived from the Greek word epigenesis which originally described the influence of genetic processes on development.2During the 1990s there became a renewed interest ingenetic assimilation. This lead to elucidation of the molecular basis of Conrad Waddingtons observations in which environmental stress caused genetic assimilation of certain phenotypic characteristics inDrosophilafruit flies. Since then, research efforts have been focused on unraveling the epigenetic mechanisms related to these types of changes.3Currently, DNA methylation is one of the most broadly studied and well-characterized epigenetic modifications dating back to studies done by Griffith and Mahler in 1969 which suggested that DNA methylation may be important in long term memory function.4Other major modifications includechromatinremodeling, histone modifications, and non-coding RNA mechanisms. The renewed interest in epigenetics has led to new findings about the relationship between epigenetic changes and a host of disorders including various cancers, mental retardation associated disorders, immune disorders, neuropsychiatric disorders and pediatric disorders.

Epigenetics: fundamentals Cancer. Cancer was the first human disease to be linked toepigenetics. Studies performed by Feinberg and Vogelstein in 1983, using primary human tumor tissues, found that genes of colorectal cancer cells were substantially hypomethylated compared with normal tissues.1DNAhypomethylationcan activate oncogenes and initiatechromosomeinstability, whereas DNAhypermethylationinitiates silencing of tumor suppressor genes. An accumulation of genetic and epigenetic errors can transform a normal cell into an invasive or metastatic tumor cell. Additionally, DNAmethylationpatterns may cause abnormal expression of cancer-associated genes. Global histone modification patterns are also found to correlate with cancers such as prostate, breast, and pancreatic cancer. Subsequently, epigenetic changes can be used as biomarkers for the molecular diagnosis of early cancer.

Mental Retardation Disorders. Epigenetic changes are also linked to several disorders that result in intellectual disabilities such as ATR-X, Fragile X, Rett, Beckwith-Weidman (BWS), Prader-Willi and Angelman syndromes..2For example, the imprint disorders Prader-Willi syndrome and Angelman syndrome, display an abnormalphenotypeas a result of the absence of the paternal or maternal copy of a gene, respectively. In these imprint disorders, there is a genetic deletion in chromosome 15 in a majority of patients. The same gene on the corresponding chromosome cannot compensate for the deletion because it has been turned off by methylation, an epigenetic modification. Genetic deletions inherited from the father result in Prader-Willi syndrome, and those inherited from the mother, Angelman syndrome.

Immunity & Related Disorders. There are several pieces of evidence showing that loss of epigenetic control over complex immune processes contributes to autoimmune disease. Abnormal DNA methylation has been observed in patients with lupus whose T cells exhibit decreased DNA methyltransferase activity and hypomethylated DNA. Disregulation of this pathway apparently leads to overexpression of methylation-sensitive genes such as the leukocyte function-associated factor (LFA1), which causes lupus-like autoimmunity. Interestingly, LFA1 expression is also required for the development of arthritis, which raises the possibility that altered DNA methylation patterns may contribute to other diseases displaying idiopathic autoimmunity.

Neuropsychiatric Disorders. Epigenetic errors also play a role in the causation of complex adult psychiatric, autistic, and neurodegenerative disorders. Several reports have associated schizophrenia and mood disorders with DNA rearrangements that include theDNMTgenes. DNMT1 is selectively overexpressed in gamma-aminobutyric acid (GABA)-ergic interneurons of schizophrenic brains, whereas hypermethylation has been shown to repress expression of Reelin (a protein required for normal neurotransmission, memory formation and synaptic plasticity) in brain tissue from patients with schizophrenia and patients with bipolar illness and psychosis. A role for aberrant methylation mediated by folate levels has been suggested as a factor in Alzheimers disease; also some preliminary evidence supports a model that incorporates both genetic and epigenetic contributions in the causation of autism. Autism has been linked to the region on chromosome 15 that is responsible for Prader-Willi syndrome and Angelman syndrome. Findings at autopsy of brain tissue from patients with autism have revealed a deficiency in MECP2 expression that appears to account for reduced expression of several relevant genes.

Pediatric Syndromes. In addition to epigenetic alterations, specific mutations affecting components of the epigenetic pathway have been identified that are responsible for several syndromes: DNMT3B in ICF (immunodeficiency, centromeric instability and facial anomalies) syndrome, MECP2 in Rett syndrome, ATRX in ATR-X syndrome (a-thalassemia/mental retardation syndrome, X-linked), and DNA repeats in facioscapulohu