Cellular molecular (DiResta section)
Cellular molecular (DiResta section) 255
Popular in Cellular Molecular (Dr. Mallery and Dr. DiResta)
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This 27 page Study Guide was uploaded by Dominique Ayala on Wednesday February 24, 2016. The Study Guide belongs to 255 at University of Miami taught by Dr. Mallery and Dr. Diresta in Fall 2015. Since its upload, it has received 88 views. For similar materials see Cellular Molecular (Dr. Mallery and Dr. DiResta) in Biology at University of Miami.
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Date Created: 02/24/16
Chapter 5 - DNA and chromosomes DNA function • Stores genetic information • Template to repair genetic information • Contains elements to regulate expression of genetic information Potential to improve genetic How do we know that DNA carries the hereditary information? Griffith showed that heat-killed, infectious bacteria can transform harmless, living bacteria into pathogenic ones. The bacterium Streptococcus pneumoniae comes in two forms that differ from one another in their microscopic appearance and in their ability to cause disease. Cells of the pathogenic strain, which are lethal when injected into mice, are encased in a slimy, glistening polysaccharide capsule. When grown on a plate of nutrients in the laboratory, this disease-causing bacterium forms colonies that look dome-shaped and smooth; hence it is designated the S form. The harmless strain of the pneumococcus, on the other hand, lacks this protective coat; it forms colonies that appear flat and rough—hence, it is referred to as the R form. As illustrated, Griffith found that a substance present in the pathogenic S strain could permanently change, or transform, the nonlethal R strain into the deadly S strain. How do we know that DNA carries the hereditary information? Avery, MacLeod, and McCarty demonstrated that DNA is the genetic material. The researchers prepared an extract from the disease-causing S strain of pneumococci and showed that the “transforming principle” that would permanently change the harmless R-strain pneumococci into the pathogenic S strain is DNA. This was the first evidence that DNA could serve as the genetic material. Hershey and Chase showed definitively that genes are made of DNA. (A) The researchers worked with T2 viruses, which are made entirely of protein and DNA. Each virus acts as a molecular syringe, injecting its genetic material into a bacterium; the empty viral capsule remains attached to the outside of the cell. (B) To determine whether the genetic material of the virus is protein or DNA, the researchers 32 radioactively labeled the DNA in one batch of35iruses with P and the proteins in a second batch of viruses with S. Because DNA lacks sulfur and the proteins lack phosphorus, these radioactive isotopes provided a handy way for the researchers to distinguish these two types of molecules. These labeled viruses were allowed to infect and replicate inside E. coli, and the mixture was then disrupted by brief pulsing in a Waring blender and separated to part the infected bacteria from the empty viral heads. When the researchers measured the radioactivity, they found that much of the 3P-labeled DNA had entered the bacterial 35 cells, while the vast majority of the S-labeled proteins remained in solution with the spent viral particles. Nucleotide chains make DNA strands Base + Sugar = Nucleoside Base + Sugar + Phosphate = Nucleotide Single DNA strands make a DNA double helix DNA is made of four nucleotide building blocks. (A) Each nucleotide is composed of a sugar–phosphate covalently linked to a base—guanine (G) in this figure. (B) The nucleotides are covalently linked together into polynucleotide chains, with a sugar– phosphate backbone from which the bases (A, C, G, and T) extend. (C) A DNA molecule is composed of two polynucleotide chains (DNA strands) held together by hydrogen bonds between the paired bases. The arrows on the DNA strands indicate the polarities of the two strands, which run antiparallel to each other in the DNA molecule. (D) Although the DNA is shown straightened out in (C), in reality, it is wound into a double helix, as shown here. Chemical directionality of a nucleic acid strand. Chemical directionality of a nucleic acid strand. Shown here are alternative representations of a single strand of DNA containing only three bases: cytosine (C), adenine (A), and guanine (G). (a) The chemical structure shows a hydroxyl group at the 3′ end and a phosphate group at the 5′ end. Note also that two phosphoester bonds link adjacent nucleotides; this two-bond linkage commonly is referred to as a phosphodiester bond. (b) In the “stick” diagram (top), the sugars are indicated as vertical lines and the phosphodiester bonds as slanting lines; the bases are denoted by their single-letter abbreviations. In the simplest representation (bottom), only the bases are indicated. By convention, a polynucleotide sequence is always written in the 5′→3′ direction (left to right) unless otherwise indicated. Hydrogen bonds and van der Waals interactions hold the DNA double helix (A) The shapes and chemical structure of the bases allow hydrogen bonds to form efficiently only between A and T and between G and C, where atoms that are able to form hydrogen bonds can be brought close together without perturbing the double helix. Two hydrogen bonds form between A and T, whereas three form between G and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel—that is, oriented in opposite directions. (B) A short section of the double helix viewed from its side. Four base pairs are shown. The nucleotides are linked together covalently by phosphodiester bonds through the 3’-hydroxyl (–OH) group of one sugar and the 5’-phosphate (–OPO )3of the next. This linkage gives each polynucleotide strand a chemical polarity; that is, its two ends are chemically different. The 3’ end carries an unlinked –OH group attached to the 3’ position on the sugar ring; the 5’ end carries a free phosphate group attached to the 5’ position on the sugar Thought Question An A-T base pair is stabilized by only two hydrogen bonds. Hydrogen-bonding schemes of very similar strengths can also be drawn between other base combinations that normally do not occur in DNA molecules, such as the A-C and the A-G pairs. What would happen if these pairs formed during DNA replication and the inappropriate bases were incorporated? Discuss why this does not often happen. Double Helix A space-filling model shows the conformation of the DNA double helix. The two DNA strands wind around each other to form a right-handed helix with 10 bases per turn. Shown here are 1.5 turns of the DNA double helix. The coiling of the two strands around each other creates two grooves in the double helix. The wider groove is called the major groove and the smaller one the minor groove. The colors of the atoms are: N, blue; O, red; P, yellow; and H, white. Double helix is flexible The conserved C-terminal domain of the TATA box–binding protein (TBP) binds to the minor groove of specific DNA sequences rich in A and T, untwisting and sharply bending the double helix. Bending of DNA is critical to the dense packing of DNA in chromatin. Protein interaction can bend DNA. Transcription of most eukaryotic genes requires participation of TBP. Nucleotide chain stability: RNA, DNA and the ribose 2’ hydroxyl group Why did DNA evolve to be the carrier of genetic information in cells as opposed to RNA? The hydrogen at the 2′ position in the deoxyribose of DNA makes it a far more stable molecule than RNA, which instead has a hydroxyl group at the 2′ position of ribose Base-catalyzed hydrolysis of RNA. The 2′-hydroxyl group in RNA can act as a nucleophile, attacking the phosphodiester bond. The 2′,3′ cyclic monophosphate derivative is further hydrolyzed to a mixture of 2′ and 3′ monophosphates. This mechanism of phosphodiester bond hydrolysis cannot occur in DNA, which lacks 2′-hydroxyl groups. G•C content of DNA affects melting temperature. *During replication and transcription of DNA, the strands of the double helix must separate to allow the internal edges of the bases to pair with the bases of the nucleotides being polymerized into new polynucleotide chains. G·C content of DNA affects melting temperature. The temperature at which DNA denatures increases with the proportion of G·C pairs. (a) Melting of doubled-stranded DNA can be monitored by the absorption of ultraviolet light at 260 nm. As regions of double- stranded DNA unpair, the absorption of light by those regions increases almost twofold. The temperature at which half the bases in a double- stranded DNA sample have denatured is denoted T (mor “temperature of melting”). Light absorption by single-stranded DNA changes much less as the temperature is increased. (b) ThemT is a function of the G·C content of the DNA; the higher the G+C percentage, the greater the Tm. Order the following oligonucleotides by the their T (from lowest to highest) when annealed to their complementary strands of DNA. Three dimensional geometry of DNA Although eukaryotic nuclear DNA is linear, long loops of DNA are fixed in place within chromosomes. Thus torsional stress and the consequent formation of supercoils also could occur during replication of nuclear DNA. Topoisomerase I in eukaryotic nuclei relieves any torsional stress in nuclear DNA that would develop in the absence of this enzyme. Most genes contain information to make proteins. Each protein-coding gene is used to produce RNA molecules, which then direct the production of the specific protein molecules. Information stored in DNA What sequences are required to maintain a DNA molecule in a cell and transmit it to its progeny? • Replication origin (Ori) • Anchor or link for segregation (Ori, centromeres) • Selection component (genes) • In linear DNA: elements to protect shrinkage of DNA during replication (Telomeres) Chromosomes • Single enormously long linear DNA molecule • Human genome: 3.2×10 nucleotides • DNA packed by proteins into chromatin Each human chromosome can be “painted” a different color to allow its unambiguous identification. The chromosomes shown here were isolated from a cell undergoing nuclear division (mitosis) and are therefore in a highly compact (condensed) state. Chromosome painting is carried out by exposing the chromosomes to a collection of human DNA molecules that have been coupled to a combination of fluorescent dyes. For example, DNA molecules derived from Chromosome 1 are labeled with one specific dye combination, those from Chromosome 2 with another, and so on. Because the labeled DNA can form base pairs (hybridize) only to its chromosome of origin,each chromosome is differently colored. For such experiments, the chromosomes are treated so that the individual strands of the double-helical DNA molecules partly separate to enable base-pairing with the labeled, single-stranded DNA, while keeping the chromosome structure relatively intact. (A) Micrograph shows the array of chromosomes as they originally spilled from the lysed cell. (B) The same chromosomes have been artificially lined up in order. In this so- called karyotype, the homologous chromosomes are numbered and arranged in pairs; the presence of a Y chromosome reveals that these chromosomes came from a male. • Homologous chromosomes: paired chromosomes, 1 maternal and 1 paternal • Non-homologous chromosomes: X and Y Gene arrangements in chromosomes Comparison of gene density in higher and lower eukaryotes. (a) In the diagram of the β-globin gene cluster on human chromosome 11, the green boxes represent exons of β-globin–related genes. Exons spliced together to form one mRNA are connected by caret-like spikes. The human β-globin gene cluster contains two pseudogenes (white); these regions are related to the functional globin-type genes but are not transcribed. Each red arrow indicates the location of an Alu sequence, an ≈300-bp noncoding repeated sequence that is abundant in the human genome. (b) In the diagram of yeast DNA from chromosome III, the green boxes indicate open reading frames. Most of these potential protein-coding sequences are functional genes without introns. Note the much higher proportion of noncoding-to-coding sequences in the human DNA than in the yeast DNA Chromosomes can be highly compacted The duplication and segregation of chromosomes occurs through an ordered cell cycle in proliferating cells. During interphase, the cell expresses many of its genes, and—during part of this phase—it duplicates chromosomes. Once chromosome duplication is complete, the cell can enter M phase, during which nuclear division, or mitosis, occurs. In mitosis, the duplicated chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from microtubules and other proteins. The condensed chromosomes are then captured by the mitotic spindle, one complete set is pulled to each end of the cell, and a nuclear envelope forms around each chromosome set. In the final step of M phase, the cell divides to produce two daughter cells. Only two different chromosomes are shown here for simplicity. Interphase chromosomes occupy their own distinct territories within the nucleus. DNA probes coupled with different fluorescent markers were used to paint individual interphase chromosomes in a human cell. Viewed in a fluorescence microscope, each interphase chromosome is seen to occupy its own discrete territory within the nucleus, rather than being mixed with the other chromosomes like spaghetti in a bowl. Note that pairs of homologous chromosomes, such as the two copies of Chromosome 9 indicated, are not generally located in the same position. How is DNA organized in cells? *Chromosomes are composed of compact DNA tightly-wound around nucleosomes. Chromosomal DNA is packaged inside microscopic nuclei with the help of nucleosomes, complexes of positively-charged histone proteins that strongly adhere to negatively-charged DNA. DNA winds around a single nucleosome, composed of eight histone proteins, 1.65 times. Nucleosomes fold up to form a 30-nanometer chromatin fiber, which forms loops averaging 300 nanometers in length. The 300 nm fibers are compressed and folded to produce a 250 nm-wide fiber, which is tightly coiled into the chromatid of a chromosome. DNA is wrapped around nucleosomes • Composed of 8 Histones, 2 copies each –H2A, H2B, H3, H4 • DNA 2 turns: 147 bp Variable linker (10-90 bp Nucleosomes contain DNA wrapped around a protein core of eight histone molecules. In a test tube, the nucleosome core particle can be released from chromatin by digestion of the linker DNA with a nuclease, which degrades the exposed DNA but not the DNA wound tightly around the nucleosome core. The DNA around each isolated nucleosome core particle can then be released and its length determined. With 147 nucleotide pairs in each fragment, the DNA wraps almost twice around each histone octamer. 30 nm fiber • Nucleosomes clamped by Histone H1 • Zig-zag ribbon • Two-start helix Levels of DNA compaction DNA packing occurs on several levels in chromosomes. This schematic drawing shows some of the levels thought to give rise to the highly condensed mitotic chromosome. The actual structures are still uncertain. Regulation of chromosome structure •Problem: Compaction vs. accessibility •Solution 1 Chromatin remodeling complexes Loosens or tightens DNA to make genes accessible or inaccessible to transcription Chromatin-remodeling complexes locally reposition the DNA wrapped around nucleosomes. (A)The complexes use energy derived from ATP hydrolysis to loosen the nucleosomal DNA and push it along the histone octamer, thereby exposing the DNA to other DNA-binding proteins. The blue stripes have been added to show how the nucleosome moves along the DNA. Many cycles of ATP hydrolysis are required to produce such a shift. (B)The repositioning of nucleosomes decondenses the chromatin in a particular chromosomal region; in other cases, it condenses the chromatin. • Solution 2 Histone modifications Loosens or tightens DNA to make genes accessible or inaccessible to transcription The structure of the nucleosome core particle, as determined by X-ray diffraction analysis, reveals how DNA is tightly wrapped around a disc-shaped histone octamer. The two strands of the DNA double helix are shown in gray. A portion of an H3 histone tail (green) can be seen extending from the nucleosome core particle, but the tails of the other histones have been truncated. Each histone can be modified by the covalent attachment of a number of different chemical groups, mainly to the tails. Histone H3, for example, can receive an acetyl group (Ac), a methyl group (M), or a phosphate group (P). The numbers denote the positions of the modified amino acids in the protein chain, with each amino acid designated by its one-letter code. Note that some positions, such as lysines (K) 9, 14, 23, and 27, can be modified in more than one way. Moreover, lysines can be modified with either one, two, or three methyl groups (not shown). Note that histone H3 contains 135 amino acids, most of which are in its globular portion (green), and that most modifications are on its N-terminal tail (orange). Histone code Different combinations of histone tail modifications can confer a specific meaning on the stretch of chromatin on which they occur, as indicated. Only a few of these “meanings” are known. Histone modification enzymes regulate chromosome condensation •Acetylation/Deacetylation –Histone Acetyl Transferase (HAT’s) –Histone De-Acetylation Complex (HDAC) •Additional proteins bind to acetylated histones and maintain chromatin in a closed state Which of the following modifications to histone H3 promote gene silencing? The two electron micrographs show nuclei of two different cell types. Can you tell from these pictures which of the two cells is transcribing more of its genes? Explain how you arrived at your answer. In the electron micrographs, you can detect chromatin regions of two different densities; the densely stained regions correspond to heterochromatin, while less condensed chromatin is more lightly stained. The chromatin in A is mostly in the form of condensed, transcriptionally inactive heterochromatin, whereas most of the chromatin in B is decondensed and therefore potentially transcriptionally active. The nucleus in A is from a reticulocyte, a red blood cell precursor, which is largely devoted to making a single protein, hemoglobin. The nucleus in B is from a lymphocyte, which is active in transcribing many different genes. Heterochromatin-specific modifications allow heterochromatin to form and to spread. These modifications attract heterochromatin-specific proteins that reproduce the same modifications on neighboring histones. In this manner, heterochromatin can spread until it encounters a barrier DNA sequence that blocks its propagation into regions of euchromatin. Generally, heterochromatin regions are flanked by DNA sequences termed boundary elements, which form fixed borders accompanied by sharp transitions in histone modification profiles. Such elements result in the precise determination of epigenetic states among closely arranged chromosome loci, even when heterochromatin protein levels change. Boundary elements prevent spread of active/silent chromatin states The discovery of position effect variegation (PEV) in the fruit fly Drosophila melanogaster in the 1930s paved the way to revealing the importance of chromatin in regulating gene expression. The white gene, which is responsible for generating red color pigment in Drosophila eyes, normally resides in the euchromatic region. However, when the white gene is placed adjacent to pericentric heterochromatin due to chromosomal rearrangement, it is variably silenced and the different expression states are clonally inherited in different cell populations, resulting in mottled eyes. Changes in chromatin structures are inherited after replication Chapter 5: Essential Concepts • Life depends on the stable storage and inheritance of genetic information • Genetic information is carried by very long DNA molecules and is encoded in the linear sequence of four nucleotides: A, T, G, and C. • Each molecule of DNA is a double helix composed of a pair of antiparallel, complementary DNA strands, which are held together by hydrogen bonds between G-C and A-T base pairs • The genetic material of a eukaryotic cell is contained in a set of chromosomes, each formed from a single, enormously long DNA molecule that contains many genes. • When a gene is expressed, part of its nucleotide sequence is tran- scribed into RNA molecules, many of which are translated into protein • The DNA that forms each eukaryotic chromosome contains, in addi- tion to genes, many replication origins, one centromere, and two telomeres. These special DNA sequences ensure that, before cell division, each chromosome can be duplicated efficiently, and that the resulting daughter chromosomes are parceled out equally to the two daughter cells • In eukaryotic chromosomes, the DNA is tightly folded by binding to a set of histone and nonhistone proteins. This complex of DNA and protein is called chromatin • Histones pack the DNA into a repeating array of DNA–protein particles called nucleosomes, which further fold up into even more compact chromatin structures. •A cell can regulate its chromatin structure—temporarily decondensing or condensing particular regions of its chromosomes—using chromatin- remodeling complexes and enzymes that covalently modify histone tails in various ways • The loosening of chromatin to a more decondensed state allows proteins involved in gene expression, DNA replication, and DNA repair to gain access to the necessary DNA sequences • Some forms of chromatin have a pattern of histone tail modification that causes the DNA to become so highly condensed that its genes cannot be expressed to produce RNA; such condensation occurs on all chromosomes during mitosis and in the heterochromatin of interphase chromosomes.
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