Bacterial Genetics
Though bacteria usually reproduce asexually, they do have parasexual processes which pass genetic info from two parents, like sexual reproduction. This includes transformation (donor DNA from the environments incorporated into recipient genome), conjugation (direct bacterial contact with DNA transference) and transduction (DNA transfer between bacteria via becteriophage). Bacteria contain one single main chromosome, along with several plasmids. Genetic exchange usually happens between a fragment of a chromosome of a donor strain and the main chromosome of a recipient strain, where recombination occurs, a unidirectional and usually nonreciprocal process. The donor strain is usually linear, requiring even numbers of exchange events, but can also be a circular plasmid. Overall, these processes are devastating, as they are responsible for adapting bacteria to antibiotics and creating highly pathogenic and hardy species of bacteria.
Bacterial tests are run by growing colonies which are scanned for mutants � these include antibiotic-resistant mutants, nutritional mutants or carbon-source mutants, as well as those based in morphology, phage resistance, etc. Prototrophic mutants are nutritionally independent, whereas auxotrophic mutants require a nutritional supplement. These mutants are found by using nonselective media, then selective media to find the missing nutrient needed. After mutants are examined, the three parasexual processes can be differentiated by two criteria: cell contact and DNase sensitivity. Transformation and transduction do not require cell contact, but conjugation does; only transformation is DNase sensitive. In the U-tube test, the presence of a filter through which only DNA and viruses can pass helps to determine if conjugation occurs � the two different strains cannot mix, so when DNase is applied, the only possibility for recombination is transduction.
Not all bacteria molecules are available for transformation: only competent cells which secrete competence factor protein can be transformed. This secretion is induced in high-density colonies that have competence pheromones, which are exuded by growing bacteria. Donor DNA binds to receptors on the cell surface and is transported into the cell, where it is hydrolyzed by exonuclease. Recombination proteins search for similar DNA-binding sequences on both the donor and recipient cell, whereupon the two strands are integrated. It is relatively nonselective in most bacteria, but certain species requires DNA only from closely related species. Afterwards, gene mapping studies can be done. The basis behind these tests is the fact that if two genes are close to each other, they will likely be on the same DNA fragment and have a higher probability of being cotransformed. So, single and double transformation frequencies are recorded versus decreasing donor DNA concentration to determine the closeness of two genes.
Conjugation usually utilizes E. coli for studies. One of the first studies with conjugation showed that gene transfer from a donor (F+) strain to a recipient strain (F-) usually turned the recipients into F+ cells, because of the transfer of the F or fertility factor within the F plasmid, made of 94K bp. Genes on this plasmid direct cell-cell contact and the synthesis of sex pili, that attach cells together. The F factor can insert into the chromosome because of the presence of short, transposable insertion sequences. Since the F factor can exist independently of the main chromosome, it is called an episome. Certain cells with a high recombination frequency of the F factor are known as Hfr cells, which vary in the location of F factor integration and direction of gene transfer. Hfr cells have the F factor integrated into the main chromosome. The F factor can be excised in Hfr cells by reversing the integration process; if it messes up, F� factor cells are made, which also transfer at high frequency into F- cells. This type of transfer is called sexduction. In conjugation genetic mapping, interrupted mating experiments are used to determine closeness of genes by differing time of entry. The map is 100 minutes long, the time for a full transfer. In extremely close genes, three point crosses are done using single mutant donor and double mutant recipients, or vice versa. One gene is used as an outside marker, as its location will have been previously ascertained, thus there are only two possibilities for order.
In generalized transduction, phages can carry any bacterial genes from one cell to another. By a packaging error, only bacterial chromosomes are packed into the phage head � this can act like any other phage, spreading the bacterial DNA snippet as �viral DNA�. It must integrate otherwise the DNA snippet will dissolve. In abortive transduction, the donor DNA is already expressed in the recipient, thus bacterial division creates two partially diploid bacteria, one expressing the donor DNA, and the other not. Specialized transduction has transducing particles with both viral and bacterial DNA. First, a temperate phage (that can either lyse the cell or enter into a lysogenic relationship) must circularize and integrate into the host chromosome, creating a prophage. Excision occurs at a loop; when this occurs aberrantly, it can sometimes include bacterial DNA. The prophage (known as low-frequency transduction lysates) then encourages lytic growth. Upon infection of another bacteria, it can integrate into host genes. If it needs help integrating, a normal phage can act as a helper phage. The chromosome containing the prophage and the helper phage is an unstable transductant because the cell can easily revert to the original phenotype, or the lytic cycle can be induced, creating lysate that is 50% of each type of phage. This lysate is known as high-frequency transduction lysate. Mapping proceeds like transformation mapping, except usually generalized transduction cells are used.
Mitochondrial and Chloroplastic Genetics
Organelle heredity is non-Mendelian, as there is an unequal biparental contribution and an irregular segregation of alleles. Genetic segregation is best displayed by color variegation in plant leaves. This is mostly caused by the presence of chloroplasts that can make chlorophyll or not; if both kinds are present, it is a heteroplasmy. Otherwise, it is a homoplasmy. Correns found that plant color was passed on by maternal inheritance through the ovules � chloroplasts were the obvious candidate. Further experiments justified the choice of chloroplast, but inheritance is through the pollen as well in certain species. Sager was able to construct the chloroplast DNA by studying recombination with antibiotic resistance, which seems to usually exhibit uniparental inheritance. Some yeast mutants form tiny colonies which have some defect in glucose metabolism. These petite mutants (as opposed to the normal grande) are divided into the neutral petite mutants who can�t pass on the phenotype, and the suppressive petite mutants who can. Suppressive petite mutants have grossly mutated mitochondrial DNA that suppresses the wild-type; neutral petite mutants don�t have any mitochondrial DNA and so pass on the grande phenotype.
Mitochondrial DNA (mtDNA) molecules are usually circular and occur in large number in each cell. Sometimes, intramolecular recombination can lead to several different circular molecules. Plant mtDNA is much larger than animal mtDNA, as it contains unassigned reading frames which have no discernible function. When animal mtDNA strands are separated, heavy and light strands are visible, which code for different proteins all the way around the molecule. Plant mtDNA tend to have more separate transcription units. A key difference to normal transcription is the fact that some codons have different meanings. As well, many mtRNA transcripts will undergo editing, or changes to the plant transcripts, with the help of guide RNAs. Trans-splicing can occur, by gathering up scattered transcripts and splicing together through intron interaction. From there, mitochondrial gene products go to act in the mitochondrion with the help of nuclear gene products like ribosomal and cytosolic proteins mtDNA mutations can lead to Leber�s hereditary optic neuropathy and Pearson marrow-pancreas syndrome, caused by an amino acid mutation and a large deletion respectively.
Chloroplasts are a specialized form of plastids, categorized with chromoplasts, amyloplasts and elaioplasts. These all contain chloroplast DNA (cpDNA). cpDNA is not as large as mtDNA and is a closed circular molecule The main species difference is the differential arrangement of the same set of genes. Chloroplasts form from a process called biogenesis, where proplastids are stimulated by light and form vesicles from an inner membrane. From these vesicles, thylakoid stacks are created, which mature into grana.
Mitochondria and chloroplasts are thought to have come about by endosymbiosis, a special case of symbiosis in which one partner lives inside the other. It is accepted that they were once free-living and somehow became incorporated into primitive eukaryotic cells. Since that beginning, significant genetic reshuffling has occurred and increased organelle dependence on nuclear gene products. Gene content within the organelle seems to have changed, as has structural variation and nucleotide sequences, in comparisons of protist and animal mtDNA.
�Genetics: An Evolutionary Mate for 'Eve�? by Boyce Rensberger, The Washington Post
About 10 years ago, molecular biologists found evidence in human genes that all people share a common female ancestor, dubbed Eve, who lived in Africa about 200,000 years ago�.Now comes corroboration from a different kind of genetic study. While the earlier claim was based on DNA transmitted only through the maternal lineage (mitochondrial DNA), the new report uses DNA transmitted and possessed only by males (the Y chromosome).Michael F. Hammer, a researcher in molecular evolution at the University of Arizona in Tucson, reported in the Nov. 23 Nature that his analysis of a part of the Y sex chromosome indicates that modern humans descended from a common male ancestor who lived 188,000 years ago. Although the new report does not say where that ancient man, whom some are calling 'Adam,' lived, his age is close enough to Eve's for this kind of work. Both analyses are based on counting mutations that distinguish a portion of one modern person's DNA from that of others and using a "molecular clock" that assumes the mutations arise at a known, constant rate. Even though the studies refer to a single man or woman in the past, they do not imply that those people were a couple or even that they were the only parents of all humans. Their primary significance is in pointing to the time when anatomically modern human beings, Homo sapiens sapiens, evolved from a more primitive ancestor, generally thought to be an archaic form of Homo sapiens.�
Cancer Genetics
Cancer is caused by cells with unregulated growth, usually sparked by the presence of carcinogens. Big masses of cancerous cells collect, known as tumors, which can spread to other areas by metastasis � some will be benign and some will be malignant. Cancer cells are usually obvious because of their unusual appearance: aneuploid, different surface proteins, disorganized structure. Within the growth cycle itself, checkpoints exist at G1, S, G2 and M, which, if read incorrectly, evade stoppage of the cycle. The cyclins and cyclin-dependent kinases (CDKs)are very important at these junctions. CDKs must phosphorylate other proteins, but they cannot function unless complexed to cyclins. The absence of cyclin ends the cell cycle; cancer causes defects in the genetic machinery that controls the levels of these complexes, causing deregulation of the cell cycle. It was established that cancer had genetic causes by the fact that cancer is clonally inherited, that viruses can induce formation of cancer, that cancer can be induced by mutagens, and that cancer tends to run in families. Later research indicated two classes of cancer genes: the oncogenes and the tumor-suppressor genes.
Oncogenes were first discovered in retroviruses, that synthesize DNA from RNA using reverse transcriptase. Genes were isolated that were determined to be the cancer-causing agent, like v-src in the Rous sarcoma virus. The different genes are involved in growth, phosphorylation and transcription � which all contribute to the cell cycle. Various proto-oncogenes (or normal cellular oncogenes) were then discovered, which were the cellular homologues of viral oncogenes, in that they had the same exon structure, but the cellular version included introns. Their presence was probably of great value to a virus, to have a gene that stimulates increased growth of its host using an integrated genome. These cellular oncogenes can be linked to a specific cancer by the transfection test, which involves adding a marker to DNA strands before transfection into normal cells, creating colonies of cancer cells. The DNA isolate should contain the specific marker if integration has happened. For example, the ras oncogene has been linked to many types of cancer. Ras product does not cause abnormal production of proteins but instead impairs GTP hydolysis, leaving the cell indefinitely on. The ras oncogene thus acts as a dominant activator. Some cancers are caused by chromosomal rearrangements, like chronic myelogenous leukemia and the Philadelphia chromosome, which is a translocation of the c-abl oncogene breakpoint on chromosome 9 onto the bcr gene breakpoint on chromosome 22.
Tumor suppressor genes are also related to cancer; though they are anti-oncogenes, cancer can only proceed if mutations block out the function of these genes (loss of function mutations). Knudsen hypothesized that two mutational hits are required to knock out the normal tumor suppressor gene function � it seems to hold true and apply to several other cancers. Within the hereditary cancers (which make up 1% of the total), the root cause is a tumor suppressor gene rather than an oncogene. Certain genes, like pRB, p53, pAPC, phMSH2 and pBRCA have been implicated in many cancers. RB is involved in cell cycle regulation; p53 is involved in apoptosis; pAPC regulates cell renewal in the large intestine; and both phMSH2 and pBRCA repair DNA;. In the case of p53, mutation prevents transcriptional activation, causing a dominant negative effect.
Hanham and Weinberg proposed six hallmarks of pathways leading to malignant cancer
1. Cancer cells acquire self-sufficiency in the signaling processes that stimulate division and growth.
2. Cancer cells are abnormally insensitive to signals that inhibit growth.
3. Cancer cells can evade programmed cell death.
4. Cancer cells can acquire limitless replicative potential.
5. Cancer cells develop ways to nourish themselves.
6. Cancer cells can acquire the ability to invade other tissues and colonize them.
The Molecular Structure of Chromosomes
The genetic code has three functions: genotypic, or replication; phenotypic, or gene expression; and evolutionary, or mutation. It is stored in chromosomes, which are compounds of proteins and nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA was proved to be the genetic material after a set of experiments. Griffith first proved that transformation existed: living type IIR cells were able to transform heat-killed type IIIS cells into living type IIIS bacteria. Later, Avery, MacCleod and McCarty showed that protease and RNase were unable to impair transformation, but DNase could, meaning that the substance in question really was DNA. Hershey and Chase offered some backup evidence by radioactively marking DNA in bacteriophage T2 and finding radioactivity in the pellet after centrifugation. Recent methods have allowed bacterial cells to be infected with pure phage DNA; these are called transfection experiments. However, it is also true that RNA can store genetic information, as RNA viruses have only RNA as genetic material, and no DNA.
Nucleic acids are made of a phosphate, a pentose, and a base. The pentose is either ribose or 2-deoxyribose, and the bases are either adenine (A), guanine (G), thymine (T), cytosine (C) or uracil (U). Adenine and guanine are double-ring purines, and the others are single-ring pyrimidines. DNA exists as a double stranded polymer, and RNA exists as a single strand. DNA was also found to exist in a double helix � Chargaff�s work suggested that thymine and adenine, as well as cytosine and guanine, were present in a fixed interrelationship, and Franklin�s X-ray diffraction patterns showed that DNA was a two-stranded structure with repeating substructures. Watson and Crick conluded that it was a right-handed double helix, coiled in a spiral, with phosphodiester bonds connecting nucleotide to nucleotide at the sugars. The two strands are held together by H-bonds, and are complementary to each other. As well, base pairs are stacked 0.34 nm apart and the sugar-phosphate backbones are antiparallel (�opposite polarity�). This form was eventually known as B-DNA, but other forms exist. A-DNA is right handed with 11 bp per turn and a helix diameter of 2.3 nm. Z-DNA is right handed with 12 bp per turn and a helix diameter of 1.8 nm. Comparatively, B-DNA is right handed with 10 bp per turn and a helix diameter of 1.9 nm. Within a chromosome, the DNA is held in a supercoil with cleaved strands that twist around each other at the end, not allowing any spin. This allows the DNA molecule to collapse into a tightly coiled structure. In most organisms, the DNA undergoes negative supercoiling, with rotation in the left-handed direction.
Prokaryotes are monoploid and usually contain all the genes within one chromosome, which contain a single molecule of either RNA or DNA. The functional state of bacterial DNA is the folded genome, similar to the nucleoid in eukaryotes. Here, the DNA molecules are segregated into negatively supercoiled domains; in bacteria, there are 50 of these domains.
Eukaryotes have much more DNA and higher ploidy stored in several chromosomes. It is packaged as chromatin, which contain basic proteins called histones (1H1, 2H2a, 2H2b, 2H3, 2H4) and acidic nonhistone chromosomal proteins. The histones remain largely constant within species, and complex in beads called nucleosomes, which are the building blocks of chromatin.
There are two models for DNA distribution within the cell: the multineme and unineme model, that describe how many DNA molecules are stretched across the chromosome. Evidence from lampbrush chromosomes shows that each chromosome has just one giant molecule of DNA. These large chromosomes form loops which break into chromatids; treatment with DNase breaks the loops but RNase does not ruin continuity, showing a unineme structure. Since DNA is sensitive to shearing � that is, on vibration, the DNA breaks easily � new methods had to be devised to study the DNA closely. Pulsed-field gel electrophoresis can separate large DNA molecules through electric fields applied perpendicular to each other; the slower reorientation and slower migration of the larger molecules shows a clear definition of single molecules. Autoradiography is used to detect and localize radioactive isotopes used for DNA labeling by exposure to a photographic emulsion that is sensitive to low-energy radiation. Autoradiographic maps show lengthy pieces of DNA whose mass would contain 2/3 of the DNA present in the largest chromosomes. Viscoelastometry analyzes the viscosity of molecules in solution � some studies have indicated correlations between total DNA and size of the largest DNA molecules.
DNA is condensed in three levels. First, it is packaged as a supercoil into nucleosomes, made of linker DNA and a nucleosome core which is invulnerable to nuclease attack. An octamer of histones (H2a, H2b, H3, H4) is involved in nuclease protection and added stability, by passing between turns of the DNA superhelix. The second level involves additional folding of the 10-nm micleosome fiber into the 30-nm chromatin fiber of meiotic chromosomes. H1 histone is involved in this level. The third level has the nonhistone chromosomal proteins forming a scaffold that further condenses the chromatin fiber into a metaphase chromosome. This seems to involve formation of independently supercoiled loops, but the mechanism is as yet unknown.
The centromere is a constricted region of the metaphase chromosome that does not seem to duplicate. In fact, replacement of one CEN by another has no detectable effect on cell division. The telomere is a region on the ends of chromosomes that reproduce differently than other parts of the chromosome. These telomeres contain TTAAGGG repeats that shorten with age � however, they do not shorten in cancerous cells. Telomeres have other functions, including preventing deoxyribonuclease digestion, preventing fusion with other DNA molecules, and facilitating replication of the chromosome ends without loss of material. Some species have movable telomeres, earning the designation transposable genetic elements. Most telomeres end with a G-rich single-stranded region of the 3� end of the DNA.
DNA has been split into three classes: 1) unique or single-copy DNA sequences, with up to 10 copies per genome, make between 30 and 85% of the eukaryotic genome, and 100% of the prokaryotic genome; 2) moderately repetitive DNA sequences, with up to 100,000 copies per genome, make up between 5 and 80% of the eukaryotic genome, are relatively heterogeneous, and made up mostly of transposable genetic elements; and 3) highly repetitive DNA sequences, with more than 100,000 copies per genome, make up between 3 and 45% of the eukaryotic genome, do not encode for proteins and are mostly present in telomeres and centromeres. The first evidence of the existence of repetitive DNA came from the discovery of satellite bands and satellite DNA that are made up of repetitive sequences. Centrifugation studies tended to find one main band of DNA and several of these satellite bands in eukaryotic species. When DNA is heated to 100 degrees, it denatures. When the temperature is cooled again, the process reverses, and sequences renature. The speed of the process relies on DNA concentration and complexity, meaning that the populous repetitive DNA will renature fastest. In the human genome, 44% of the genome is made from 4 classes of repetitive DNA: Long Interspersed Nuclear Elements (LINE), Short Interspersed Nuclear Elements (SINE), transposable elements containing Long Terminal Repeats (LTRs) and DNA tranposons.
DNA Replication
DNA synthesis happens incredibly fast and very accurately, and like anything else, involves initiation, elongation and termination. Watson and Crick put forth a model of replication known as semiconservative replication. That is, each strand of DNA is a separate template and can direct synthesis of a new complementary strand by base-pairing. Thus, the parental double helix can be half-conserved, or semi-conserved. Other possible mechanisms include conservative replication, with the double helix being entirely conserved and acting as the full template, or dispersive replication, which includes synthesis and rejoining of segments from both parental and progeny strands. Meselsohn and Stahl, by observing DNA replication with �heavy� and �light� strands showed that progeny were half heavy and half light, the exact proportion predicted by semiconservative replication. They were able to do this by equilibrium density-gradient centrifugation, a method to separate molecules of different density. Taylor, Woods and Hughes were able to witness the same physically by radiation staining of bean cells without the ability to undergo anaphase separation, retaining all chromosomes.
Cairns found that unwinding of the two complementary parental strands and semiconservative replication occurred almost simultaneously, and that Y-shaped structures developed during the middle of replication. These structures were replication forks that practiced bidirectional replication (though some species do replicate unidirectionally). This established the existence of an origin of replication, which controls the replication of a replicon. In the prokaryotes, only one replicon exist, but eukaryotes contain many, meaning multiple replication origins. One repeated sequence within the origin is rich in A:T and facilitates the formation of a replication bubble. The formation of this localized zone of denaturation is an essential first step in replication. The multiple origins as a whole appear to be specific sequences, called Autonomously Replicating Sequences (ARS). Any base-pair changes removes the origin of replication function.
DNA polymerase I or Kornberg�s enzyme catalyzes the covalent addition of nucleotides to preexisting DNA chains. Its function requires the presence of dATP, dTTP, dGTP, dCTP and pre-existing DNA in the presence of Mg2+ ions. The pre-existing DNA serves as a primer and as a template. Since the polymerase cannot synthesize new chains from scratch, it requires a free hydroxyl group on which to attach nucleotides with phosphodiester bonds. Of course, the new chain must be formed from the template DNA strand. The direction of synthesis is always 5� to 3�. As well, DNA polymerase I also conducts exonuclease activity in both directions (degradation starting at the ends). There are four other DNA polymerases. DNA polymerase II is a DNA repair enzyme with 5� to 3� polymerase activity and 3� to 5� exonuclease activity. DNA polymerase III has the same activities but only has 5� to 3� exonuclease activity on single-stranded DNA. DNA polymerase IV and V are important in replication of damaged DNA. In eukaryotes, there are at least seven different DNA polymerases, which I will call 1 through 7. 2, 6 and 7 are DNA repair enzymes; 1, 4 and 5 work with semiconservative replication, and 3 is responsible for mitochondrial DNA replication. Some of these lack the 3� to 5� exonuclease activity present in prokaryotic versions. The prokaryotic DNA polymerase III was found to be the true DNA replicase responsible for semiconservative replication. It is a multimeric enzyme that has 3 subunits in its core that clamps the DNA and allows it to slide while remaining tethered. The 3� to 5� exonuclease proofreads the DNA � this is DNA polymerase III(e), 3, 4 and 5.
The DNA strands being copied have opposite chemical polarity � how is this paradox resolved? Continuous synthesis of the leading strand (5�t o 3�) and discontinuous synthesis of the lagging strand (3� to 5�). The small fragments of DNA from the lagging strand are called Okazaki fragments, and are connected together using DNA ligase. This enzyme only fills in when there are missing phosphodiester linkages and no missing bases. Mostly, NAD is used as a cofactor, but sometimes ATP is used. Since new fragments must be created all the time on the lagging strand, priming is constantly needed. This can be done using RNA primers made by DNA primase. These primers are short but provide the 3� hydroxyl group necessary. On the lagging strand, Okazaki fragment synthesis is terminated by DNA polymerase III when the fragment bumps into the RNA primers of the next fragment. DNA polymerase I then gets rid of the RNA primers with its 5� to 3� exonuclease activity. It subsequently patches it up with a DNA chain using its 5� to 3� polymerase activity. The unwinding process (which requires a 3000 rev/min spin) is catalyzed by DNA helicases. The DNA strands are kept in that extended single-stranded form by single-strand DNA-binding (SSB) protein. Its high cooperativity allows quick coating of the entire region of DNA. Then, the required axes of rotation is maintained by DNA topoisomerases. DNA topoisomerase I produces temporary single-strand breaks (removing supercoils one at a time) and DNA topoisomerase II produces transient double-strand breaks in DNA (removing and introducing supercoils two at a time). DNA gyrase, the best known DNA toposiomerase II, is important for introducing negative supercoils in bacterial chromosomes; it folds across the molecule, cleaves both strands, passes the helix through the break and reseals the break.
In the circular prokaryotic chromosome, the replication bubble is formed by the interaction of prepriming proteins (DnaA, DnaB and DnaC) with oriC. This initiation complex contributes to the formation of two bidirectional replication forks. RNA primers synthesized by DNA primase initiate synthesis of new strands: one is required for the leading strand, but several are needed for the laggin strands. Initiation of Okazaki fragments are carried out by the primosome, a complex of DNA primase and helicase. Its ATP-powered movements unwind the double helix, make RNA primers, break DNA, replace the primers, seal the DNA and then condense it back into the nucleoid by DNA gyrase action, all in one concerted motion. The whole apparatus is called the replisome, containing the DNA polymerase III holoenzyme. Viruses, bacteria and amphibians use rolling-circle replication; here, one strand remains intact and rolls while serving as a template. It can produce either single or double-stranded progeny DNA.
In eukaryotic systems, there are some key differences in chromosome replication. During the cell cycle, replication only occurs during the S phase, and frequently involves multiple replicons per chromosome, as replication at one origin would take much too long. Sometimes, 2 or more polymerases can work at a single replication fork, and nucleosomes may be duplicated at the same point.. Finally, telomeres are attached to new cells by an RNA-containing enzyme called telomerase, which is being targeted in possible cancer treatments.
DNA Transcription
Metabolism (the reactions occurring in living organisms collectively) occurs by sequences of enzyme-catalyzed reactions, with each enzyme specified by one or more genes. Therefore, each step in a metabolic pathway is under genetic control. DNA transfers information to RNA through the process of transcription, and is a reversible step; translation of RNA to protein is irreversible. During transcription, one DNA strand is used as a template to synthesize a strand of RNA � this is known as the gene transcript. The primary transcript is usually equivalent to the mRNA molecule in prokaryotes. In eukaryotes, primary transcripts are precursors to mRNAs and are called pre-mRNAs. These have noncoding introns that separate the coded exons. Eventaully, spliceosomes will removes the intron sequences. Eventually, four forms of RNA will be produced: mRNA, tRNA, rRNA and snRNA, which make up the spliceosomes. Transcription happens within the nucleus and the product RNAs are processed before they travel into the cytoplasm to work.
RNA synthesis is similar to DNA synthesis, except it uses ribonucleoside triphosphates, only one strand of DNA as template for the complementary RNA chain, uracil takes the place of thymine and synthesis can be initiated without a primer strand. The RNA strand is known as sense strands, as they make sense in coding for proteins. The complementary RNA to the mRNA is known as antisense RNA. Like DNA chains, synthesis occurs in the 5� to 3� direction. RNA polymerases initiates transcription at promoter sites, creating a transcription bubble. In prokaryotes, there is only one RNA polymerase involved with the transcription unit and transcription process. This holoenzyme has a tetrameric core harboring the RnTP and DNA template binding regions. The alpha subunit is involved in assembling the core, and the sigma factor is just involved in initiation of transcription. The sigma factor binds to the -35 or recognition sequence on the DNA. Short chains of up to 9 ribonucleotides are synthesized and released until chains of more than 10 can be produced, whereupon RNA polymerase will continue down the DNA to the downstream sequences. RNA polymerase unwinds the two strands during the process, with help from the -10 sequence on the promoter. As the nucleotides are produced, phosphodiester bonds are created to hold the nucleotides together. After sigma is released, elongation occurs with continual unwinding and rewinding behind the polymerase pathway � the transcription bubble is about 18 nucleotide pairs long at any given moment. Termination occurs when RNA polymerase encounters a termination signal. It will be either rho-dependent or rho-independent termination. Often, translation and degradation will occur at the same time as transcription, since they all work in the 5� to 3� direction.
In eukaryotes, the population of primary transcripts is called heterogeneous nuclear RNA (hnRNA). Transcription utilizes RNA polymerases I, II and III, which transcribe the synthesis of different RNA � generally I does rRNA, II does nuclear pre-mRNA, and III does tRNA and snRNA. With pre-mRNA synthesis, RNA polymerase II recognizes short conserved elements near the transcription start site. First comes the TATA box with sequence TATAAAA; and then the CAAT box, with sequence GGCCAATCT; followed by the GC box and octamer box, or GGGCGG and ATTGCAT. Basal transcription factors (TFIIX) are required. TFIID must bind first to interact with TATA, and then TFIIA and TFIIB. TFIIF catalyzes unwinding, and TFIIE helps out in the actual transcription. Early in elongation, 7-methyl guanosine caps are added to the 5� ends. These help protect the chain from nuclease degradation. After elongation, endonucleolytic cleavage cuts the 3� end whereupon a poly (A) polymerase will add a poly(A) tail, called polyadenylation. The formation of the tail requires a specificity component to direct this reaction. Termination requires discrete signals in polymerase I and III, unlike with polymerase II. RNA editing can then occur before translation starts, to change individual bases or inserting and deleting uridine monophosphate residues. It is mediated by guide RNAs when done in mitochondria. The role and requirements of editing are not yet clear.
When DNA is hybridized with RNA, R-loops are created, where RNA has displaced DNA to form a complex. With pre-transcription and post-transcription comparison of R-loops, Leder was able to prove the existence of introns. Genes have differing numbers of introns: some have large continuous introns, and some have several smaller introns. To remove introns, RNA splicing must occur with dead-on accuracy. The conserved sequence in studies seems to be the TACTAAC box, but it is rather poorly conserved. In any case, there are three mechanisms: endonucleolytic cleavage and ligation reactions; autocatalysis; and spliceosome reaction. In the first mechanism, a splicing endonuclease makes two precise cuts at each intron end, before a splicing ligase joins the two halves. Autocatalysis is a self-splicing activity mediated by the RNA molecule itself, requiring no energy source or protein. It does use GTP as a cofactor and a monovalent and divalent cation. The intron is then removed by two phosphoester bond transfers, and then the introns can then circularize using another phosphoester bond transfer. This reaction is dependent on the secondary structure. In spliceosome splicing, the five snRNAs (U1, U2, U4, U5, U6) exist in RNA-protein complexes called snRNPs, with each having an individual snRNP and U4 and U6 being present together in a fourth. Spliceosomes are made from all 4 of these snRNPs. First, a phosphoester linkage is cleaved at the 5� intron splice site (where U1 is bound) and a phosphodiester linkage is created between the 5� G of the intron and the conserved A on the 3� end of the intron (where U2, U5, and U4/U6 are bound). The 3� introns splice site is then cleaved and the two exons joined by a phosphodiester linkage.
Protein Translation
Proteins make up 15% of the wet weight of cells and are the most prevalent component of living organisms. These proteins are made of polypeptides that consist of amino acids linked together by covalent bonds. All of these amino acids except proline contain a free amino group and a free carboxyl group; they differ by the side groups present: hydrophobic (nonpolar), hydrophilic (polar), acidic (- charge) or basic (+ charge). The number of possible polypeptides given 20 different amino acids is huge: 20x. The amino acids within the peptide are connected via peptide bonds, which form between the amino group of one and the carboxyl of the other, with the elimination of a water molecule. The structure of a protein exists on four different levels: primary, the amino acid sequence; secondary, the spatial interrelationships of amino acids; tertiary, with its overall folding in 3D space; and quaternary, referring to the association of polypeptides in a multimeric protein. Protein folding is usually spontaneous according to amino acid structure but can require use of chaperone proteins. The secondary structure exists most commonly as alpha helices or beta sheets. Alpha helices have peptide bonds H-bonded to each other, and beta sheets have multiple folds back in on itself which are held place by H-bonds. The tertiary conformations are maintained with hydrophilic side toward the surface. Here, disulfide covalent bonds, ionic bonds, H-bonds, hydrophobic interactions and Van der Waals interactions help hold the protein in place. Ionic bonds connect opposing charges, H-bonds connect electronegative atoms to H, hydrophobic interactions are nonpolar group associations and Vander Walls is a proximity attraction.
Translation occurs with the participation of over 50 polypeptides and 4 RNA molecules per ribosome, 20 amino acid-activating enzymes, 40-60 different tRNA molecules and proteins involved in chain initiation, elongation and termination. Before translation begins, genetic information must be transferred to mRNA intermediaries. Translation can then occur once mRNA, rRNA and tRNA adaptor molecules have been gathered together at the ribosomes. Ribosomes are located in the cytoplasm an contain a smaller 30S subunit and a larger 50S subunit. These contain 4 rRNA, which are transcribed in the nucleolus and made by cleavage with exoribonuclease. Their genes are present in thousands of copies, usually in tandem arrays in the nucleolar organizer regions of the chromosomes. Several ribosomes simultaneously translate an mRNA, forming a polyribosome. At initiation, mRNA forms a complex with the 30S subunit and initiation factor IF-3s, before the 50S subunit attaches and makes a 70S ribosome. Meanwhile, the amino acids are attached to the tRNA by a reaction involving aminoacyl-tRNA synthetases and ATP, which forms an amino acid-tRNA complex and AMP. Synthesis is begun by a special tRNA, tRNAfMet in response to the initiation codon, which attaches to the A or aminoacyl site on the ribosome (for incoming) with the help of initiation factor IF-2 and GTP. The tRNA codons ar e known as anticodons. After the 50 S subunit binds, the initiator tRNA moves to the P or peptidyl site (where the peptide is extended). In eukaryotes, the process requires a cap-binding protein which complexes with several initiation factors and the small 40S subunit. It then looks for an AUG in the 5� to 3� direction, before the large 60S subunit binds and the initiation factors dissociate. Elongation can then occur. Elongation factor EF-Tu with GTP is required for binding to the A site. After usage it is inactive until it is rephosphorylated by elongation factor EF-Rs. Peptidyl transferase moves the amino acid chain to the tRNA in the A site from the P site. Elongation factor EF-G and GTP then move what is in the A site to the P site and what is in the P site to the E site and so on. Elongation continues until termination codons are recognized by special release factors (eRF), which releases the mRNA and dissociate the ribosome into its subunits.
The genetic code is comma-free, degenerate, nearly universal, ordered, non-overlapping, made of triplets and contains start and stop codons. Crick was first to establish the existence of a triplet code, meaning 3 nucleotides per codon, by studying the changing of the reading frame by base-pair additions and deletions with mutagens. Eventually, Nirenberg and Matthaei were able to discover the different codons and what they stood for. AUG, or Met, was found to be the initiator. UAA, UAG and UGA were found to be terminators. The code was termed degenerate for the occurrence of more than one codon per amino acid, except for Met and Trp.
The degeneracy of the genetic code allows more than one tRNA to exist for certain amino acids and more than one codon to be recognized by tRNA. The first two bases of the codon follow strict base-pairing rule, but the third follows less stringent rules. Crick called this wobble; by this hypothesis, G can recognize either U or C on the codon, C can recognize G, A can recognize U, U can recognize A or G, and I can recognize A, U or G. I stands for inosine, which is a post-transcriptional modification of adenosine and only exists on tRNA. Suppressor mutations can produce altered codon recognition in tRNA. The amber mutation is where a mutation produces the UAG termination codon, truncating the protein. Generally, termination mutations are called nonsense mutations, and missense mutations specify a different amino acid all together. Suppression of nonsense mutations occur in the tRNA � so these are referred to as suppressor tRNA. Much of this has been verified by comparison of nucleotide and amino acid sequences in vitro.
Mutation, DNA Repair and Recombination
Mutation is the source for genetic variability in evolution. It refers both to the actual change and the process by which the change comes about, ending with a new phenotype labeled as a mutant. We usually refer to point mutations as mutations, which include substitution, or insertion or deletions. DNA repair enzymes exist to repair the damage caused by point mutations. Mutations may occur in any cell at any stage, and can be divided between germinal mutations and somatic mutations. Germinal mutations get passed into the progeny, while somatic mutations only pass to the descendants of that specific cell. Mutations can also be divided into spontaneous and induced mutations. Mutagens cause induced mutations and increase mutation rate by orders of magnitude. The connection was first proved by Muller, when he showed that X-rays could induce recessive lethals on the X chromosome of Drosophila. Mutation was thought of for a long time under Lamarckian terms, but the Lederberg�s replica plating methods provided evidence that mutation was random and nonadaptive. With replica plating, bacterial cultures were transferred into sterile plates with streptomycin by the use of velvet. Some colonies were able to grow, showing existence of streptomycin resistance before exposure to the antibiotic. Mutation was also found to be reversible. Forward mutations refers to the initial mutation; reverse mutation restores the original phenotype by back mutation at the same site or by the occurrence of a suppressor mutation elsewhere that compensates for the first mutation.
Mutations produce many phenotypic differences. Isoalleles (neutral mutations) have little effect on phenotype; null alleles produce nonfunctional or no gene products; and recessive lethals are mutations in genes necessary for growth. Mutation can be either dominant or recessive, and in most cases with phenotypic effects, the mutation is recessive and deleterious. In genetic studies, conditional lethal mutations are very helpful, especially in determining morphogenesis: in one environment, the mutation is lethal (restrictive condition) and in another, it is viable (permissive condition). The main classes of conditional lethals are auxotrophic mutants (unable to synthesize an essential metabolite); temperature-sensitive mutants and suppressor-sensitive mutants (requires a suppressor present for viability).
Tautomeric shifts � the movement of hydrogen atoms from one position in a base to another � changes pairing potential, and if the base has shifted at the moment of replication, a mutation will exist. If the changes are between pyrimidines or between purines, it is called a transitions; otherwise, it is a transversion. Mutations involving additions or deletions are called frameshift mutations because they alter the reading frames of the base-pair triplets. Chemical mutagens can be divided into those that are mutagenic to replicating and non-replicating DNA, like alkylating agents, hydroxylating agents and nitrous acid (add alkyl, -OH and amino groups), and those that are mutagenic only to replicating DNA, like base analogs. Base analogs are similar to the normal bases and can be incorporated, but they increase frequency of mispairing and mutation during replication. These chemical mutagens can also act as carcinogens, which was confirmed by the Ames test, which compared frequency of reversion in chemical to spontaneous reversion without. Ionizing radiation (X-rays, gamma rays) and nonionizing radiation (UV light) causes release of ions or electron excitation, increasing reactivity. Movement of transposable genetic elements, or transposons, can also cause mutation. Random insertion will render some genes nonfunctional. A type of mutation associated with human disease is the prevalence of simple tandem repeats. Trinucleotide repeats are probably the worst offender, causing many inherited diseases in humans, like Huntington�s. The phenomenon of anticipation is the increasing severity of the disease as the trinucleotide copy number increases.
DNA repair mechanisms include light-dependent repair, excision repair, mismatch repair, postreplication repair and error-prone repair system (SOS response). Light-dependent repair, or photoreactivation, isn�t used much in mammals as most mammalian cells don�t have access to light. In bacteria, it uses a light-activated enzyme called DNA photolyase which splits thymine, cytosine and cytosine-thymine dimers when they crosslink. Excision repair has a DNA repair endonuclease recognizing, binding to and excising the damaged base before a DNA polymerase fills in the gap by using the untouched strand as template. DNA ligase then seals the breakage left. Base excision repair systems remove abnormal bases as initiated by DNA glycosylases, while nucleotide excision repair removes the larger defects using excinuclease. Nucleotide repair uses many more proteins. Mismatch repairs are a kind of proofreading that ensures correctly matched nucleotides. Postreplication repair occurs when both original strands have been lost. The repairs are done through a recombination-dependent repair process, mediated by the recA protein. The SOS response occurs as a last-ditch response when damaged by UV light. A whole battery of DNA repair, recombination and replication occurs in which any gaps are filled in opposite damaged strands as a last resort, though the frequency of error is high. The importance of these pathways is underlined when viewing inherited human disorders that result from defects in the repair mechanisms.
DNA recombination involves cleavage and rejoining of DNA molecules. The full picture of recombination has evolved by study of recombination-deficient mutants that don�t possess all the proteins necessary for crossing over. Endonuclease cleaves single strands of each of the parent molecules and then these are displaced by DNA helicase. In E. coli, this is run by the RecBCD complex. The strands than exchange pairing partners, stimulated by proteins like recA.. Single strand assimilation occurs, whereby a strand of DNA displaces its homolog in a double helix, first from the first to the second, then the second to the first.DNA ligase then recombines the cleaved strands. If the breaks don�t occur at the same place, endonuclease and polymerase can repair it. This then produces an X-shaped chi form which will then break to produce two recombinant DNA molecules. A double-strand break model has also been proposed. Gene conversion can occur during recombination. Its effects seem like that caused by a mutation, except that it occurs at higher frequencies and always produces the allele on the homologous chromosome. It is caused some time during repair synthesis.