Biology 240 Exam 3 Study Guide!
Biology 240 chapter 9 Notes!
-With advancing research, extranuclear inheritance is a recognized aspect of genetics.
Organelle heredity: DNA contained in mitochondria/chloroplasts determines the phenotypic characteristics of the offspring. Ex: uniparental transmission usually is from the female through the egg to progeny.
Infectious heredity: symbiotic or parasitic association with the microorganism. The phenotype is affected in the cytoplasm of the host cells. We also discuss several other topics like Inflation is an increase in the overall, what?
Maternal effect: nuclear gene products are stored in the egg and transmitted through the cytoplasm of the offspring. Gene products are distributed to cells of the developing embryo and influence the phenotype.
-What do they share in common?---information is transmitted through the cytoplasm, not the nucleus---extranuclear inheritance.
-Analysis of mutant alleles from chloroplasts/mitochondria is difficult because the function of organelles is dependent on gene products from nuclear/organelle DNA. Also, the organelles contributed to each progeny cell follows cell division. Heteroplasmy: variation of genetic content in organelles.
-Carl Correns provided evidence linking inheritance by chloroplast transmission in Mirabilis jalapa--with green and white leaves. The white leaves did not have chlorophyll. Inheritance of the chloroplast is determined in the cytoplasm of the maternal parent because pollen (contributes little cytoplasm) has no effect on the zygote.
-Chloroplast mutations in Chlamydomonas--has single large chloroplast with 75 circular DNA copies. DNA that contributes resistance to antibiotics was passed onto the female parent (mt+). The genes depended on the mother. Linked to transmission of the chloroplast. When mating fuses both for the zygote, genes are derived from the mother---inheritance is uniparental. But mt- (the father) determines the mitochondrial information.
If you want to learn more check out Is axo-axonic terminal and retrograde actions are the same?
-Mitochondrial mutations are transmitted through the cytoplasm during reproduction. Ex: poky mutant in Neurospora crassa (bread mold).Slow growth is associated with the poky mutation and impaired mitochondrial function (due to lack of cytochrome proteins essential for electron transport). The strain is maternally inherited. Another example is yeast and mutational variants from mitochondria--If you want to learn more check out Seedless vascular plants is?
deficiency in cellular respiration due to abnormal ETC. Creates petites that become facultative anaerobes to survive. Segregational petites exhibit Mendelian inheritance, neutrals yield only wild type and suppressives give rise to diploid zygotes that after meiosis become haploid for poky (petite). Petite cells that inherit both wild and poky mitochondria express dominant-negative mutations, which suppress the wild type function.
-A possible explanation suggests mutant (or deleted) DNA in mitochondria (mtDNA) replicates more rapidly, resulting in mutant DNA taking over or dominating the phenotype by numbers alone. Another suggestion is that recombination between mutant and wild types brings errors to the normal mtDNA. Don't forget about the age old question of What is the monotonic sequences?
Endosymbiotic theory: theory that mitochondria and chloroplasts arose independently about 2 billion years ago as free-living bacteria. The cells were engulfed by primitive eukaryotic cells and entered a symbiotic relationship. The bacteria could no longer live autonomously and the eukaryotic host gained the ability to perform oxidative respiration or photosynthesis.
-During the evolution of host cells, the symbiont bacteria also underwent independent changes. The primary alteration was the transfer of genes from the bacterium to the host cell nucleus. The products are now encoded and transcribed in the nucleus and translated into the cytoplasm before moving into the organelle. DNA in mitochondria is tiny compared to ancient bacteria. Similar occurred in chloroplasts. We also discuss several other topics like When a standard deviation of a data set is 0 then?
-Chloroplasts have 2 functions: Capture light energy and split water. CO2 is fixed into sugars.
-Chloroplast DNA (cpDNA) is fairly uniform among different organisms from 100-225 kb in length. Similar to DNA in prokarya. Circular, double stranded, no proteins. Size of cpDNA is larger than mtDNA---the difference is due to the long noncoding sequences between and within genes (introns). cpDNA encodes numerous tRNAs and ribosomal proteins specific to chloroplast ribosomes---similar to bacterial.
-For mtDNA, introns are usually absent from the mitochondrial genes, and repetitions are not usually present. Human mtDNA encodes 2 rRNAs and 22 tRNAs and 13 polypeptides essential to respiration of the organelle. In vertebrate DNA, the 2 strands vary in density. The majority of proteins functioning in the mitochondria are encoded by nuclear genes (ex: DNA and RNA polymerases, initiating and elongating factors…etc.). Distinct form the cytoplasmic counterparts even though they are encoded in the same place.
-mtDNA is susceptible to mutations because 1. It does not have protection form mutations provided by histone proteins present in nuclear DNA, 2. DNA repair for mtDNA is limited, and 3. Mitochondria concentrate highly mutagenic reactive
oxygen species (ROS) generated by respiration--in confined space it is highly toxic and can damage proteins, lipids and mtDNA.
In order for a human disorder to be attributed to mutant altered mitochondria: We also discuss several other topics like How do you calculate marginal rate of substitution?
1. Inheritance must exhibit a maternal pattern.
2. The disorder must reflect the deficiency in bioenergetics function of the organelle.
3. There must be a mutation in 1 or more of the mitochondrial genes. Today, there are over 150 syndromes related to mitochondrial dysfunction.
Myclonic epilepsy and ragged-red fiber disease (MERRF): demonstrates maternal inheritance and offspring of affected fathers are normal. The affected express ataxia, deafness, dementia and epilepsy. Related to a mutation in one of the twenty two mtDNA encoding tRNAs. Reveals patterns of heteroplasmy. Without heteroplasmy, the mutation would be lethal in the affected.
Leber’s hereditary optic neuropathy (LHON): the affected exhibit maternal inheritance and mtDNA lesions. Characterized by sudden blindness. Average age for this is 27due to disruption of oxidative phosphorylation. No family history is usually involved; tends to be sporadic.
Kearns-Sayre syndrome (KSS): loss of vision, hearing and heart conditions. Involves deletions of mtDNA.
-mitochondrial disorders also seem to play a role in almost all major conditions, including type II diabetes, autism, atherosclerosis, infertility, Parkinson’s, Alzheimer’s, Huntington’s and some cancers. Also involved in aging due to a decrease in ATP production for cells or deletions/mutations in mtDNA.
-Transplants of nuclear genomes can now prevent mtDNA mutations for offspring, eliminating the risk for disorders. Involves the rhesus monkey genome implanted in the egg of the mother and is fertilized--mitochondrial swapping. (Still pending legal approval).
**In maternal effect, the genotype of the female parent, not the embryo, determines the phenotype of the offspring.
Possible Exam questions:
-Inheritance of green and white leaves in Mirabilis jalapa is due to maternal influence: FALSE
-Shell coiling in Lymnea peregra is influenced by_____ due to: maternal effect; orientation of the spindle apparatus early in cleavage.
-One explanation for organelle inheritance is that: mitochondria and chloroplasts are subject to DNA mutation
-Future nuclear transplantation might be available to treat mtDNA disorders. This is known as: mitochondrial swapping.
Biology 240 Chapter 10 Notes:
Genetic material: information contained in genes that when passed onto a new generation, influences the form and characteristics of each individual. Also a source of variability through mutation.
-For a molecule to serve as genetic material, must have 4 characteristics: replication, storage of information, expression of information, variation by mutation. Replication is fundamental for all living organisms. Once doubled, must be partitioned equally to all cells. Also occurs in formation of gametes. Storage of information requires the molecules to act as repositories of genetic information that may/may not be expressed. The need to have vast information is needed to produce varying gene products. Expression is a complex process that is the underlying basis for the concept of information flow within the cell. The initial event is transcription of DNA, where mRNA, tRNA and rRNA are synthesized. mRNA is translated to protein by mediation of tRNA and rRNA. Each mRNA is specific to the gene it’s a product of. Translation converts mRNA to protein (from amino acid chains). Transcription, translation, DNA to RNA to protein--central dogma of molecular genetics.
-DNA first studied in 1869 by Swiss chemist Miescher. Isolated nuclei and derived acidic substance with DNA--nuclein. 1910, Levene learned DNA contained nucleotides. Started basis for tetranucleotide hypothesis for DNA structure. In the 1940s, Chargaff elaborated on this, demonstrating the variation of nucleotides in different organisms.
-1944, Avery, McLeod, and McCarty published “transforming principle” in bacteria. Based of Griffith’s 1927 experiment on pneumonia. Smooth strains are infectious and have a polysaccharide capsule to protect themselves from phagocytic bacteria; rough strains do not. IIR and IIIS used in experiment. Heat killed smooth strain did not result in in death of mice, but the combination of rough plus heat-killed smooth strain resulted in death because of some interaction (transformation). A transforming principle might be part of the polysaccharide capsule or compound required for capsule synthesis. The smooth cells served as a nutrient source for rough strains. Dawson in 1931 took one step further and showed that smooth could incubate in rough to regain infectious ability.
-Avery McLeod and McCarty isolated smooth pneumonia strains and centrifuged, collected and heat-killed them. They then obtained a filtrate that retained the ability to transform rough strains. The protein was removed and polysaccharides then digested. The transforming principle was found to be DNA because a phosphorous ratio was found. After eliminating all proteins with digesting enzyme ribonuclease (RNase), the transforming ability was still retained. When DNase was injected, the transforming ability was lost.
-The study of E. coli and bacteriophage T2 revealed that DNA is carried in the phage head to replicate and infect host cells. Experiment by Hershey and Chase labeled DNA with phosphorous and protein with sulfur. After being centrifuged, bacteriophages were checked to see that DNA was transferred to bacterial cells after adsorption. This demonstrated that genetic material phage T2 was DNA, not protein (found outside cell).
-Spizizer and Fraser independently reported that by using protoplasts (enzymatically naked cells), they could initiate phage reproduction with disrupted T2 particles. Virus did not have to be intact for infection.
Transfection: infection process by only viral nucleic acid; proves conclusively that OX174 phages’ DNA alone contains all the necessary information for production of mature viruses.
Indirect evidence that supports DNA as the Genetic material in Eukarya:
-Found in nucleus (along with protein). Mitochondria and chloroplasts have DNA with genetic roles. Protein is found everywhere.
-The amount of DNA in haploid sperm to the diploid number in blood cells and the number of chromosomes correlates.
UV light is capable of inducing mutations. Action spectrum of UV light measures the wavelength by the number of mutations it induces. The absorption spectrum is compared with this to tell what could be the genetic material. ** The molecule serving as the genetic material is expected to absorb at the wavelength(s) found to be mutagenic (DNA and RNA at 260 um, protein at 280).
-The strongest evidence of DNA as the genetic material is with recombinant DNA technology. Segments of eukaryotic DNA are spliced into bacterial DNA; the complex is inserted in the bacterial cell and the gene expression is monitored. If a eukaryotic gene is introduced, the subsequent protein is produced.
-Some viruses contain RNA and not a DNA core (RNA is the genetic material). When purified RNA from tobacco mosaic virus (TMV) is spread on tobacco leaves, characteristic lesions caused by viral infection subsequently appear (RNA is the genetic material). Spiegelman/Pace demonstrated that RNA can be isolated and replicated in vitro, depending on the enzyme RNA replicase, which is isolated from
host E. coli cells following infection. When replicated RNA is added to E. coli, transfection occurs. RNA directs viral reproduction.
Retroviruses use RNa as a templateto synthesize DNA via reverse transcription under direction of reverse transcriptase.
Nucleotides: building blocks of nucleic acids. Consist of a nitrogenous base, pentose sugar and a phosphate group. 2 type of nitrogenous bases: purines (A, G) and pyrimidines (C,T). Pentose sugars can be ribose (for RNA) or deoxyribose (for DNA).
-The data available to Watson and Crick allowed them to propose the double helix and came from: 1.base decomposition analysis of hydrolyzed samples of DNA (from Chargaff) and 2. X-ray diffraction studies of DNA (from Rosalind Franklin) (1953).
1. Amount of A is proportional to T; amount of G is proportional to C 2. The sum of purines (A+G) equals the sum of pyrimidines (C+T) 3. The percentage of (G+C) doesn’t necessarily equal (A+T)
DNA Model by Watson and Crick (semiconservative):
1. 2 long polynucleotide chains coiled around a central axis (double helix) 2. Both chains antiparallel (3’-5’ and 5’-3’)
3. Bases of both chains flat, stacked.
4. Paired nitrogenous bases (A-T, G-C) Complimentary with hydrogen bonding 5. Each turn is 10 base pairs long
6. Major groove alternating with a minor groove.
7. Hydrophobic interior of double helix, hydrophilic phosphate backbone
-Structure of RNA chemically similar to DNA, but single stranded. (Complimentary copies of DNA)
-Telomerase RNA and RNA primer is involved in DAN replication at end of the chromosome.
Possible Exam Questions:
1. If protein had been the hereditary material, what would have Hershey and Chase’s experiment resulted in?: P inside the cell and not S.
2. Sample DNA is 27% guanine, so what percent thymine is in the sample?: 23% 27% is cytosine, 100-54=46 46/2= 23% adenine, 23% thymine
Biology 240 Chapter 11 Notes!
Semiconservative replication: involves the synthesis of a new strand using the complementation of an old one.
Conservative replication: complimentary polynucleotide chains are synthesized, following newly created strands that come together and parents reassociate.
Dispersive replication: parental strands are dispersed into two new double helices following replication. Each strand has both old and new DNA. Involves cleavage of parental strands during replication. Most complex of the three theories.
1958-Meselson-Stahl experiment-growing E. coli cells for many generations, labeling N as the source of cell nutrients. N 15 is more dense than N 14. Almost all N containing molecules had N 15. Sedimentation equilibrium centrifugation used to separate N 15 from N 14/ N 15 was transferred to N 14, making all new synthesis of DNA with N 14. Cells replicate over generations. After one generation; isolated DNA fell in middle of centrifuge (density intermediate- semiconservative replication). After 2 cell divisions, DNA had one intermediate and one lighter band (N 14). Proportion of lighter band increased over cell divisions.
If DNA has dispersive replication, all generations after t=0 would be intermediate. In each successive generation, N 15/N 14 would decrease and the band would become lighter (not observed).
-Experiment with broad bean in vila faba (1957-Hughes, Taylor, Woods) observed semi-conservative replication. Root tips were monitored for replication and labeled DNA with H-thymidine and autoradiography: where applied cytologically, pinpoints location of radioisotopes in a cell. Dark grains identify newly synthesized DNA. Arrested cells in metaphase after one generation. After one replication, both sister chromatids were radioactive with one old and one new strand. After second replication in an unlabeled medium, only one of the two are radioactive because half of parents strands were unlabeled.
-At each point along the chromosome where replication is occurring, strands of helix unwound and a replication fork was created. Initially appears at a point of origin of synthesis and moves along DNA duplex as replication proceeds. If replication is bidirectional, 2 forks are present, migrating opposite ways: Replication is the length of DNA that is being replicated following one initiation event.
-In E. coli, OriC is the single origin where replication is initiated **(characteristic of bacteria because their chromosome is circular). The entire chromosome is a replicon, and replication is bidirectional.
DNA Polymerase I: directs DNA synthesis. Needed for all 4 dNTPs and template DNA. Precursor dNTP contains 3 phosphate groups attached to 5’ carbon. As 2 terminal phosphates are cleaved in synthesis, the last phosphate attached to 5’ carbon is
linked to 3’ OH--chain of elongation in 5’--3’ direction by the addition of 1 nucleotide at a time to the 3’ end.
DNA Pol I, II and III cannot initiate DNA synthesis, but can elongate an existing DNA strand (primer) and have the ability to polymerize in one direction, pause, reverse and excise in the other direction. Pol I is present in greater amounts and can be more stable. Removes primers during synthesis and fills primer gaps.
Holoenzyme: active form of DNA Pol III; α,ε ,θ = core enzyme; impacts the
catalytic function of the holoenzyme. Sliding clamp leader pairs with the core enzyme to facilitate the function of the sliding DNA clamp--dependent on ATP hydrolysis. Links to core enzyme, shapes as donut to encircle unreplicated DNA, leads way in synthesis.
Processivity: the length of DNA replicated by the core enzyme before detaching from the template. One sliding clamp exists for one core enzyme.
Ori C in E. coli is the origin of replication (AT-rich site (less stable, enhances helical unwinding)) DNaA initiator protein is responsible for initiating replication by binding to a region on 9mers at Ori C. Undergoes change and associates with 13mers to destabilize the helix and expose single stranded DNA (ssDNA)--binding DNA helicase. Binds to replication fork, initiates replication, opens up DNA helix. Single stranded binding proteins (SSBs) bind to single strands of DNA to form a template. Supercoiling tension occurs and coiling tension is created ahead of the replication fork. DNA gyrase (one of the topoisomerases) relaxes this tension.
Synthesis of DNA begins with a type of RNA. A short segment of RNA is synthesized by the DNA template through primase, which is recruited to the replication fork by DNA helicase. DNA Pol III added deoxy ribonucleotides, initiating DNA synthesis. Later, an RNA primer is replaced by DNA under Pol I.
DNA Pol III synthesizes DNA in only the 5’--3’ direction; the other strand of DNA is 3’--5’. Only 1 strand serves as the template for continuous DNA synthesis (leading strand). As the replication fork progresses, many points of initiation are necessary
on the opposite side of the template, resulting in discontinuous DNA synthesis of the lagging strand. Okazaki fragments are small fragments of newly synthesized DNA that include RNA primers, converted into longer and longer DNA strands of higher molecular weight as synthesis proceeds. Discontinuous synthesis requires enzymes to remove primers and unite Okazaki fragments into the lagging strand. DNA Pol I removes primers and DNA ligase joins the fragments.
-DNA is A-T rich because of less H bonds--easier to separate for DNA synthesis
-Both strands of DNA are synthesized simultaneously, using one core enzyme for each. Lagging strand forms a loop to do this. A monomer of the enzyme will find the
completed Okazaki fragment and release the lagging strand. A new loop is created, etc. To check for DNA errors, DNA Pol I has exonuclease activity (3’---5’) (Proofreading).
-Ligase deficient and proofreading deficient mutations can be lethal; genetic analysis often uses conditional mutations, expressed under one condition but not another (ex: temperature sensitive mutations)--not expressed at a permissive temperature, but at a restrictive temperature is expressed with the mutant phenotype.
Differences between Eukaryotic and Prokaryotic replication:
Eukarya deal with more DNA. Polymerases synthesize DNA 25x slower. Multiple replication origins contained (replication bubbles). Origins in yeast (autonomously replicating sequences (ARSs)) consist of about 120 base pairs containing a consensus sequence (nearly the same in all ARSs) of 11 base pairs. A pre-replication complex (pre-RC) controls timing of replication at replication origins. In G1, replication origins are recognized by the origin recognition complex (ORC) that tags the site as the initiation of replication. Once synthesis begins, pre-RC disassembles until the next G1 phase. Distinguishes sites that have already been replicated. Cell cycle kinases are essential at the initiation site to trigger proteins at the S phase. The End result: DNA unwinding, stabilization, DNA Pols at origins, initiation of synthesis.
-To accommodate the number of replicons, eukarya have more DNA Pol molecules than bacteria. Also use a larger number of different DNA Pol types (about 14 different types).
Pol α enzyme synthesizes RNA primers on the leading and lagging strands. Also
adds 10-20 complimentary deoxyribonucleotides. Contains low processivity (strength of association between enzyme and substrate; the length of DNA synthesized before dissociating). Once the primer is in place, polymerase switching occurs, where Pol α dissociates and is replaced by Pol δ∨ε; extends primers
on opposite strands of DNA, has more processivity, exonuclease activity, proofreads. Pol ε is on the leading strand, Pol δ on the lagging. These also do some DNA repair and recombination. All 3 are essential for viability.
-eukarya have Okazaki fragments 10x smaller than prokarya.
-One of the major differences between prokaryotic/eukaryotic DNA; DNA is eukarya with DNA-binding proteins, chromatin. Consists of nucleosomes wrapped around histones. Before synthesis, nucleosomes must be modified to allow passage of
replication proteins. The nucleosome patterns are quickly reestablished for the new duplexes. Old histones assemble with new ones ahead of the replication fork. New nucleosomes are formed behind the replication fork. Carried out by chromatin assembly factors (CAFs).
-Structured differences in chromosomes also exist. Eukaryotic chromosomes are linear. Problems arise: double stranded ends of DNA molecules at the ends of linear chromosomes resemble double-stranded breasks (DSBs) that can occur when a chromosome becomes broken because of DNA damage. Double-stranded loose ends can then fuse, resulting in fusions and translocations because new DNA cannot be synthesized at the tips of single-stranded 5’ ends. Telomeres are linear chromosomes that help preserves chromosomes by creating inert ends to protect from degradation.
-A 3’ strand G-rich strand is present on one of the two DNA strands making up each telomere, and 5’ strand C-rich strand (complimentary). G-rich 3/ end lies in overhand, becoming single stranded. This allows it to create loops (t-loops) based on G-G bands. Makes DNA resistant to degradation.
-The problem with the 5’ end strand is that therea are no free 3’-OH groups available to allow synthesis of DNA. Gaps remain then on the newDNA strands at each successive round of synthesis, shortening double stranded ends of the chromosome by the length of the RNA primer. ** With each successive replication, shortening in each daughter cell becomes more severe, eventually extending beyond the telomere and potentially deleting gene-coding regions. The solution: telomerase: capable of adding more repeats to the G-rich strand (5’--3’ synthesis). As a ribonucleoprotein, contains a piece of RNA that guides attachment and serves as the template for DNA synthesis (reverse transcription). TERC and TERT aid in this. The RNA enzyme base pairs with the overhang and extends beyond; reverse transcriptase occurs, extending the G-rich lagging strand. Then regular synthesis occurs. The same happens to the C-rich strand. Telomerase is not usually active in somatic cells, however.
Homologous recombination occurs at equivalent positions along 2 chromosomes with substantial DNA homology.
Possible exam questions:
1. Difference between functions of DNA Pol I and Pol III: DNA Polymerase III is the main polymerase of the replisome, whereas DNA Polymerase I removes primers and fills in the gaps that are left.
2. Which protein is responsible for unwinding DNA for replication?: DNA Helicase
Biology 240 Chapter 12 Notes!
-Prokaryotic chromosomes lack in associated proteins and have less genetic information. Prokarya can exist as single stranded, double stranded, linear or circular chromosomes and have RNA or DNA as their genetic material. Circularity is not an absolute requirement for virus replication. One thing in common with prokarya and eukarya is the ability to pack long DNA into a small volume. Bacterial chromosomes consist of a double stranded DNa molecules compacted into a nucleoid. Several DNA binding proteins are associated with this, HU and HNS (histone-like structuring) proteins. Small but abundant, these contain high percentages of positiviley charged amino acids functioning to fold and bond DNA. HNS also regulates gene activity in a nonspecific way.
Supercoiled DNA: characteristic of closed circular molecules. First observed in polyoma virus in mice. Closed-circular molecules are more compact and sediment more rapidly than linear DNA molecules but have the same molecular weight. Supercoiled shape is caused by higher energy stabilizing the double helix. In most virsues and bacteria, circular closed DNA is slightly underwound.
Topoisomers are 2 otherwise identical molecules that differ only in linking number. Enzymes that cut the closed ends of topoisomers are topoisomerases. Topoisomerase I serves to reduce the number of negative supercoils. Topoisomerase II introduces negative supercoils in DNA. This is also found in Eukarya. In both, DNA replication/transcription creates supercoils downstream as the double helix unwinds and becomes accessible to the appropriate enzyme.
Polytene chromosomes are found in the salivary glands of flies. Can be seen in the nuclei of interphase cells. Exhibit alternating band pattern, distinctive for each chromosome, called a chromomere. Polytene chromosomes represent paired homologs. Large due to repeated DNA strands. DNA of paired homologs undergo extensive replication ** but no strand separation or cytoplasmic division. Each band represents an individual gene (uncoiled during genetic activity, forming puffs).
Puffs illustrate high levels of gene activity (transcription that produces RNA). Bands that don’t turn into puffs have low levels of activity.
Lampbrush chromosome resembles brushes (discovered by Flemming). Characteristic of most vertebrate oocytes as well as spermatocytes of some insects (are meiotic chromosomes). Most experimental ones done from amphibian oocytes. Isolated in diplotene stage of prophase I, active directing metabolic activities of the developing cell. Synapsed pairs are held by chiasmata, do not condense and are usually extended. Later in meiosis, they revert to normal length (usually uncoiled).
Condensed areas of chromomeres contain lateral loops. Each meiotic chromosome composed of a pair of sister chromatids.
Chromatin: decondensed chromosomes, usually present during interphase. While in interphase, Chromatin is dispersed throughout the nucleus. As the cell enters S phase, DNA is replicated and chromatin coils and condenses.
Eukaryotic chromatin has a substantial amount of protein associated with chromosomal DNA in all cell cycle phases. Can either be positively charged histones or less positively charged nonhistone proteins. Histones contain large numbers of amino acids lysine, arginine, making possible to bond electrostatically to negative phosphate groups of nucleotides. Histones play an important role in chromatin structure; control regulating diffraction rings.
Chromatin is composed of nucleosomes. Formation of nucleosome represents the first level of packing. Characteristic of uncoiled chromatin in stacked nucleosomes from Histone I. In mitotic transition, chromatin is compacted into looped domains; coiled chromatin fibers the constitute the arms of a chromatid.
**When present in several levels of compaction within the chromatin fiber, DNA inaccessible to the interaction with other DNA binding proteins. To accommodate DNA protein interactions, chromatin must change structure (chromatin remodeling). To allow replication and gene expression, chromatin must relax and expose DNA to the proteins. There are unstructured histone tails that are not packed into histone domains with nucleosomes, but protrude from it. Other make connections with other nucleosomes. Provide potential targets along the chromatin fiber for a variety of chemical modificators that may be linked to genetic function.
Acetylation for enzyme histone acetyltransferase involves the addition of an acetyl group to the positively charged amino group on the side chain of lysine and neutralizes the positive charge. High levels of acetylation remodel chromatin fiber to increase the regions of active genes and decreases inactive regions. Methylation/phosphorylation, resulting from enzymes methyltransferase and kinase. Added to lysine and arginine, involving unfolding and condensation during and after DNA replication. Methylation within nucleosomes often correlated with gene activity in eukarya; methylation of cytosine within polynucleotides of DNA forms 5-methyl cytosine, negatively correlated with gene activity (methylation can have a positive or negative effect).
Euchromatin: parts of the chromosome that is uncoiled.
Heterochromatin: parts of the chromosome that remain condensed. Heterochromatin are generally inactive because they lack genes or contain genes that are expressed. Heterochromatin replicates later during the S phase of the cell cycle than euchromatin. Ex: telomere, centromere---help maintain structural stability. In some cases, characteristic to genetic material of eukarya, whole
chromosomes can be heterochromatic (inactivated X chromosomes Barr body). If some heterochromatic areas are translocated to new site, genetically active sites may become inert if adjacent to the heterochromatin; position effect. Chromosome bonding techniques stain chromosomes to make them distinguishable from one another. C-bonding refers to staining of only centromeric regions of mitotic chromosomes (specific area of heterochromatin). G-bonds produced by staining technique that runs along the length of each chromosome. Involves digestion of mitotic chromosomes with trypsin enzyme. Reflect heterozygosity and complexity of chromosome. Repetitive DNA: functional genes present in more than one copy (multiple-copy genes), satellite DNA (only in eukarya): makes up a variable proportion of DNA (differs slightly in density), short and repetitive, located in heterochromatic regions, flanking centromeres.
-Separation of homologs during mitosis/meiosis depends on centromeres, primary constrictions along eukaryotic chromosomes. Repetitive DNA within is critical to role (CEN Region). DNA binds a platform of proteins including the kinetochore that binds to microtubules of spindle fiber during division. Each centromere serves identical function (most DNA sequences are almost identical). Alphoid family mainly found in centromere regions and is transcribed into RNA that ultimately serves a kinetochore function.
Moderately repetitive DNA: most characteristic for tandem repeats or noncoding sequences. Ex: variable number tandem repeats (VNTRs) dispersed throughout the genome, known better as minisatellites. Allows for DNA fingerprinting for forensic study. Microsatellites or short tandem repeats (STRs) are also dispersed throughout genome and vary among individuals. Serve as molecular markers.
Transposable sequences: mobile, can potentially move to different locations in the genome; much of human genome is comprised of these.
Short interspersed elements are a set of closely related sequences, Alu family, sometimes transcribed into RNA, although the role is not clear. Long interspersed elements have sequences from the R1 family (retrotransposons). There is a large amount of DNA that appears to be noncoding; pseudogenes represent evolutionary vestiges of duplicated copies of genes that have undergone significant mutational alteration. Usually not transcribed. **Only a very small part of the genome actually codes for proteins.
Possible Exam Questions:
1. The term “satellite DNA” refers to: highly repetitive DNA in Eukaryotes, DNA fraction that separates from the bulk of the DNA during centrifugation, and DNA in the heterochromatic centromere regions of eukaryotes
2. In contrast with euchromatin, heterochromatin contains more genes and is earlier replicating: False
Biology 240 Chapter 13 Notes!
1. Genetic code is linear, using letters as the ribonucleotide base that composes mRNA, derived from DNA.
2. Each word of mRNA, referred as a triplet code, a codon (3 bases) specifies one amino acid.
3. The code is unambiguous, codes only one amino acid.
4. The code is degenerate; a given amino can be specified by more than one codon.
5. 1 start codon, 3 stop codons; triplets initiate and terminate translation. 6. Nonoverlapping; after translation any one ribonucleotide of mRNA is part of only one triplet.
7. Sequence is collinear; sequence of amino acids making up the protein. 8. Code is nearly universal (almost the same for everyone).
An insertion of a single nucleotide causes the reading frame (sequence of nucleus encoding polypeptide) to shift, changing all subsequent codons--frameshift mutations. Sequence of protein altered radically. If one or two insertions/deletions, protein cannot be produced (original frame not established). 3 insertions/deletions reestablishes frame.
Nonsense codons: blank codons. If found during translation, causes protein synthesis to stop/terminate (no protein made).
-With Nirenberg and Matthei; a cell free protein-synthesizing system created and amino acids incorporated into polypeptides. Begins with in vitro mixture with all the necessary items; ribosomes, tRNAs, amino acids, etc. (essential to translation). To trace protein synthesis, amino acids are made radioactive, DNA is synthesized. ** Probability of insertion of a specific ribonucleotide is proportional to the availability of that molecule to other ribonucleotides. RNA homopolymers are synthesized (RNA molecule with only one type of ribonucleotide (AAA,UUU,GGG)). Used to determine what amino acids resulted. RNA heteropolymers were then used to predict following combinations that made amino acids (based on proportions of ribonucleoside diphosphate)---used to predict triple codons. Triplet binding assay was used later on for specific assignments of triplet codons, taking advantage of ribosome bonds that are made with RNA. tRNA is attracted to a complimentary RNA sequence, (tRNA triplet sequence is the anticodon).
A radioactive amino acid was combined with tRNA, making it ‘charged’. Nirenberg/Matthei found out that way what sequences corresponded to what amino acids.
-The genetic code is degenerate; almost all amino acids are specified by 2,3, or 4 different codons. A pattern of degeneracy exists; sets of codons specifying the same amino acid are grouped so that the first two letters are the same; with only the third
differing (wobble hypothesis). Initial two ribonucleotides are more critical than the third. H bonding at the third position is less spatially constrained and provides more flexibility for base pairing at the third base. As a result, only about 30 different tRNAs are needed to accommodate 61 codons for an amino acid.
Ordered genetic code: chemically similar amino acids often share one or two middle bases in the different triples encoding them. Buffers the potential effect of mutation on protein function. While mutations in the second base of triplet codons result in a
change of amino acids, the chance is to one of similar chemical properties. The protein function may not be noticeably altered.
-In bacteria, initiation of translation begins with fmet (AUG)-sometimes known as the initiator codon. Formyl is usually removed after protein synthesis or methionine as a whole is removed. In eukarya, methionine alone is the initial amino acid of polypeptide synthesis. May be cleared afterward. 3 other codons (UAG, UAA, UGA) serve as termination codons--not recognized by tRNA, which causes the end of translation. Mutations that produce premature stop codons create partial polypeptides---nonsense mutation.
-There are exceptions to the universal genetic code, coding for other proteins within different organisms.
-There is the possibility that a single mRNA may have multiple initiation points for translation (overlapping genes). Gene is considered an open reading frame--DNA sequence that produces an RNA that has a start and stop codon and triplet codons specifying amino acids in between. Multiple initiation points have been observed in 0X174 phage. Optimizes the limited amount of DNA present; the disadvantage is that it is possible to have mutations in more than one protein (and can be lethal). Applies also to mammals.
Transcription: process from DNA template to RNA molecules. Results in mRNA molecule complimentary to gene sequence of one of two strands of the double helix. Each codon is then complimentary to the anticodon of tRNA, which inserts the correct amino acid into the polypeptide chain during translation. Transcription is the initial step in the process of information flow within the cell.
-the amount of RNA is generally proportional to the amount of protein in a cell; following synthesis, RNA migrates to the cytoplasm from the nucleus, where protein synthesis occurs. RNA polymerase directs synthesis of RNA without a need for a primer to initiate. NTPs serve as substrates for the enzyme, which catalyzes the polymerization of nucleoside monophosphates or nucleotides into a polynucleotide chain. Nucleotides are linked during synthesis by 5’---3’ phosphate bonds. Energy of the triphosphate cleavage supports the reaction. The active form of RNA polymerase is a holoenzyme which contains its subunits; beta and beta’ provides the catalytic mechanism and active site for transcription. Sigma factor plays a regulatory function in the initiation of RNA transcription. There is a single form of
the enzyme but multiple sigma factors (such as in E. coli), creating variations of the holoenzyme. Eukaryotes have distinct forms of RNA polymerase, each consisting of a greater number of polypeptide subunits than in bacteria.
Transcription in bacteria:
Template binding (from DNA) occurs; the site is established when RNA Pol or subunit recognizes the DNA promotors. Located in the 5’ region, **upstream from the initial point of transcription. The helix is then denatured to make DNA accessible. The point where transcription begins is the transcription start site. Consensus sequences of DNA, TATA box and TTGACA is critical to transcription. Sequences in regions adjacent to the gene are cis-acting elements--located on the same DNA molecule. Trans-acting factors are molecules that bind to these DNA elements. The degree of RNA Pol binding to different promoters varies greatly, causing variable gene expression. Mutations in promotors severely reduced initiation of gene expression. Once RA Pol binds to the initiations site, synthesizing begins in 5’--3’ direction (in terms of nascent RNA). After ribonucleotides are added to the growing RNA chain, the sigma subunit dissociates, chain elongation proceeds under the core enzyme.
RNA Pol can proofread as it adds each nucleotide, able to back up and correct mistakes. Runs until termination signal; important because of close proximity to an adjacent gene. The unique sequence of nucleotides in the termination region causes newly the newly formed transcript to fold into a hairpin secondary structure held by H bonds. Important to termination and also dependent on the termination factor rho (ρ¿ . Physically interacts with the RNA transcript to ease termination of
transcription. When RNA transcript releases from the template, the core enzyme dissociated. In bacteria, groups of genes whose products are involved in the same pathway are clustered together on the same chromosomes; genes are contiguous, large mRNA is produced and encodes more than one protein. Genes in phages are sometimes referred to as complementation groups, where RNA is called polycistronic mRNA. All proteins needed are produced at the same time.
Transcription in Eukarya:
1. Transcription occurs within the nucleus under the direction of 3 separate forms of RNA Pol; RNA transcript is not free to associate with other ribosomes prior to completion of transcription. Must move into the cytoplasm first.
2. Initiation requires compact chromatin fiber characterized by nucleosome coiling to be uncoiled and DNA to be made accessible to RNA Pol and other regulatory proteins (chromatin remodeling).
3. Eukaryotic RNA Pol relies on transcription factors to scan and bind to DNA. Enhancers and silencers may be located in the 5’ regulatory region upstream of initiation, within or downstream.
4. mRNA processing must occurs before translation can occur, involving the addition of a 5’ cap and a 3’ tail. Pre-mRNAs (hnRNAs) are usually larger than the mature mRNA. Only 25% of hnRNA is produced to mature mRNA.
RNA Pol II is responsible for initial template binding, depends on cis elements and trans-transposon factors. The first of cis acting--core promoter determines where RNA II binds to DNA and where copying begins. The proximal promoter elements, silencers and enhancers influence the rate of transcription initiation. TATA box is
also cis-acting, located upstream of the start. Enhancers/silences act in response to the requirement of gene product (how much needed). Transcription factors (trans acting)---include general transcription factors (essential-always required(TFIIB,TFIID,TFIIA---bind to TATA)) and transcriptional activators and repressors that influence the rate of RNA Pol II initiation. With binding of GFFs; TFIIP, RNA Pol II---pre-initiation complex.
-Promotor DNA positions itself over the 2 subunits of RNA Pol II, securing the duplex, called the clamp. The small duplex region of DNA separates with the enzyme and is referred as the active center; template scanned for the start site. RNA synthesis is initiated and transcription is repeated a number of times before stable RNA:DNA hybrid transcript is formed and synthesis can continue. Once the termination signal is received, the complex becomes unstable and the clamp opens, releasing DNA and RNA. Process goes from unstable to stable to unstable again.
The 7-mg cap is made during posttranslational modification and is placed at the 5’ cap, stabilizing mRNA and protecting form nuclease degradation; also facilitates transport to the cytoplasm and translation. Poly A sequence is added after the 3’ end of initial transcript, protects from degradation.
Intervening sequences are represented in the initial mRNA transcripts as expressed introns. Expressed and retained mRNA consists of exons. Splicing removes the unnecessary information from mRNA to produce functional proteins. Heteroduplexes are the result of hybridization of nucleic acids that are not perfectly complimentary; introns are present in DNA but absent in mRNA as they loop out and remain unpaired.
-Few eukaryotic genes lack introns.
-In splicing, endonucleases cleave the ends of introns and allow RNA ligase to seal the gap between exons (true in tRNAs of bacteria). Ribozymes perform self-excision as the intron serves as the source of enzymatic activity necessary for removal (Group 1 introns).
In higher eukaryotes, splicing occurs by the mediated spliceosome, containing small nuclear RNAs that can be complexed with proteins to form small nuclear ribonucleoproteins. Occurs within the nucleus. Several cases are know where introns are present in mRNAs derives from the same gene spliced in a different way
(alternative splicing), yields similar but variable mRNAs that result in protein isoforms.
Possible Exam Questions:
1. Which of the following is a ribozyme catalyzed process? (Options: exon splicing, lariat formation during pre-mRNA splicing, transcription elongation or all of the above): Exon splicing and lariat formation during pre-mRNA splicing
2. After hnRNA is processed in eukaryotes it moves through the nuclear membrane to the cytoplasm: True
Biology 240 Chapter 14 Notes!
Translation: biological polymerization of amino acids into polypeptide chains. Occurs only in association with ribosomes, which are the workbenches of this process.
tRNAs are a class of molecules that adapt to genetic information presented as codons and pairs with complementary anticodons to produce an amino acid chain during translation.
Ribosome consists of 2 subunits, one large and one small. Contain an array of ribosomal proteins. When both associated, become a monosome. In prokaryotes, ribosome is a 70S particles (S for Svedberg coefficient) and eukaryotic is 80S.
rRNA genes called rDNA, part of moderately repetitive DNA category and present in clusters in many chromosome sites. Each cluster consists of tandem repeats; each unit separated by spacer DNA (in humans, near ends of chromosomes).
Cloverleaf model of tRNA: has characteristic secondary structure based on base pairing. Resembles cloverleaf. Loops contain modified bases that do not generally pair (anticodon loop). 3’ end of tRNA contains CpA (adenosine residue), 5’pG contains guanine. 3’ end is the acceptor region, where the amino acid is linked.
Before translation proceeds, tRNA is charged by becoming chemically linked to its specific amino acid under enzymes aminoacyl tRNA synthetases. Amino acid reacted with ATP to create aminoacyladenylic acid. Covalent linkage formed between 5’phosphate group of ATP and carboxyl end of amino acid. Occurs with synthetase enzyme, forming complex that binds to specific tRNA. Amino acid transferred to 3’ end (where adenine residue located). Charged tRNA then participates in protein synthesis.
Initiation factors bind to the small subunit and attract the mRNA molecule, binding to a sit before the AUG start codon (Shine-Dalgarno sequence). In E. coli, Initiating Factor 1 blocks the A site of the small subunit of the ribosome from binding to the tRNA and IF 3 inhibits the small unit from associating with the large one. Stabilized
in P site, the charged tRNA pairs with start to set the reading frame. Upon release of IF 3, the complex forms and translation begins.
In elongation, the second tRNA enters the ribosome A site to pair with a codon, transported by one of the elongation factors. The terminal amino acid in the P site forms a covalent bond with the amino acid of the A site to start a polypeptide chain (elongation). Uncharged tRNA moves to the E site where it is ejected and to be recharged by another specific amino acid. The process repeats until a stop codon is reached from mRNA and the polypeptide is formed. Terminal codon signals action of GTP-dependent release factor, which stimulates hydrolysis of the polypeptide from the tRNA, leading to release. Then, ribosome dissociates.
Polyribosomes occur with mRNA that has already passed through one ribosome and begins to associate with another (occurs only in bacteria).
In eukaryotes, translation occurs on larger ribosomes whose rRNA and protein are more complex. Prokaryotic and eukaryotic RNA share core sequence, but in eukaryotes, lengthened by expansion sequences, which add to functionality. In eukaryotes, transcription and translation are separate (cannot be coupled). Transcription is in nucleus and translation is in cytoplasm. 5’ end of mRNA is capped with 7-mg unlike prokaryotes, must be processed before translation can occur. Kozak sequence analogous to Shine-Dalgarno in prokaryotes, as it is found upstream in eukaryotes. Poly A tail on the 3’ end of mature mRNA (prevents degradation by nucleases). Eukaryotic mRNA longer lived, greater number of elongation factors used; overall eukaryotes are more complex for translation.
Physician Garrod looked into research of alkaptonuria, disruption in the metabolic pathway of of alkapton, leads to darkening of cartilage and if not careful, leads to arthritic condition. Genetic in nature, found mostly in consanguineous relationships.
Phenylketonuria (PKU) also metabolic disorder, can result in mental retardation (autosomal recessive). Afflicted individuals unable to convert phenylalanine into tyrosine. Reaction catalyzed by enzyme phenylalanine hydroxylase, inactive in affected individuals. Accumulated phenyl. Converts to acid, badly absorbed, affects brain levels of balance.
Beadle and Tatum provided first convincing experiment confirming genes directly responsible for producing enzymes (now known as one gene one polypeptide). Worked with Neurospora and induced mutations to watch how they could grow on different mediums. None of the mutants could grow on minimal medium because unable to synthesize certain proteins due to damaged genes.
**Nearly all enzymes are proteins, but not all proteins are enzymes. Beadle-Tatum hypothesis modified to one gene one polypeptide hypothesis. First proof came from studying sickle cell anemia. Due to low oxygen tension that causes hemoglobin to become elongated. Beet demonstrated the effect of dosage compensation in heterozygotes (sickle-cell trait). Those not completely affected experience some symptoms but still had function of the hemoglobin (due to availability of gene product). Experimented with starch gel electrophoresis. Fingerprinting technique allowed analysis of amino acid composition and digestion of protein into fragments. Establishes that a single gene codes for a specific polypeptide.
Order of nucleotides corresponds to the order of amino acids in a polypeptide-- collinearity.
Four levels of protein structure:
Primary structure: specified by sequence of deoxyribonucleotides in DNA through an mRNA intermediate. Helps determine specific characteristics of higher orders of organization as a protein is formed.
Secondary structure: regular or repeating configurations in space assumed by amino acids lying close to one another in the polypeptide chain. Ex: alpha helix in DNA. Proteins can have mixt of alpha and beta pleated sheet structures.
Tertiary structures: three dimensional spatial conformation of the chain as whole.
Quaternary structure: applies only to those that have more than one polypeptide chain.
Proteins in different roles:
Hemoglobin and myoglobin are involved in oxygen, essential for cellular metabolism Collagen and keratin associated with skin, connective tissue; structural components
Actin and myosin are contractile proteins involved in muscle tissue; tubulin in function of microtubules during meiosis and spindle fibers.
Transport proteins involved in movement of molecules along the cell membrane