DNA Flashcards

Question

yeasts

Answer 1

* Simple Eukaryotes * Model for fundamental studies of eukaryote biology S. cerevisiae (most commonly studied yeast): 12 million bp DNA (6,000 genes) Distinct eukaryotic features: - membrane-enclosed nucleus, - genomic DNA arranged in 16 linear chromosomes, - subcellular organelles in cytoplasm Fast doubling time Mutants have expanded understanding of: - DNA replication - Transcription - RNA processing - Protein sorting - Regulation of cell division

Answer 2

* Relatively simple, multicellular eukaryote * 100 million bp DNA (19,000 genes)

Answer 3

The fruit fly D. melanogaster has been a crucial model organism in developmental biology 180 million bp DNA (14,000 genes) Easily maintained and bred in the laboratory Short reproductive cycle (2 weeks) – genetic experiments Embryos develop outside Short lifespan (monitor development) Advances in understanding the molecular mechanisms in animal development – body plan of complex organisms Parallels between genes in Drosophilia and vertebrates Wound healing, tissue regeneration, drug discovery, genetic disease, Parkinson’s disease research, neurodevelopment

Answer 4

* Relatively simple organism: suitable model organism for study of plant molecular biology and development * 125 million bp DNA (26,000 genes, unique 15,000) * Easily grown and genetically manipulated in the laboratory * Used to elucidate mechanisms such as flower development in higher plants

Answer 5

■ Take adult skin biopsy ■ Grow cells in tissue culture ■ Treat with special cocktail of chemicals to reprogram ■ Cells forget they were skin cells and revert to basic stem cells ■ Can be programmed into any cell type

Answer 6

■ First immortalised human cell line. ■ The oldest and most commonly used human cell line in scientific research. ■ Derived from cervical cancer cells taken on February 8, 1951, from named Henrietta Lacks, a 31-year-old African-American mother of five, who died of cancer on October 4, 1951 ■ HeLa cells have an active version of telomerase during cell division, which copies telomeres over and over again ■ Prevents the incremental shortening of telomeres that is implicated in aging and eventual cell death ■ Results in unlimited cell division and immortality

Answer 7

* About 26 of the 92 elements are considered essential for life * Carbon, hydrogen, oxygen and nitrogen make up 96% of living matter * Most of the remaining 4% consists of calcium, phosphorus, potassium and sulfur * Implications for amino acids and thus protein composition

Answer 8

Weak intermolecular interactions between partial charges on polar molecules Collectively forms a strong bond; individually can form and break easily as they are individually relatively weak bonds Often represented as dashed or dotted lines e.g. H-bonds between certain amino acid side chains and between C=O and N-H of amino acids in alpha helices and beta sheets

Answer 9

* Interactions between non-polar amino acid side chains. * Allow even non-polar groups to form favourable electrostatic interactions with one another. * Non-polar bonds can have a transient polarity and induce a transient polarity on a neighboring non-polar bond. * VdW forces are especially important in the hydrophobic interior of a protein.

Answer 10

* Not a force per se, however considered the major driving force for the folding of globular proteins. * Hydrophobic interactions cluster hydrophobic groups away from water (think of oil and water not mixing). * Non-polar amino acids have a strong tendency to cluster together, away from water, allowing very favourable H-bonds to form between water and hydrophilic amino acid residues

Answer 11

Single units = Monomers A few units joined together = Oligomers A large number of monomers joined = Polymers

Answer 12

* Principle information molecules of the cell * Two main types: DNA and RNA DNA: Deoxyribonucleic acid - The genetic material of all life (except some viruses) RNA: Ribonucleic acids - Messenger RNA (mRNA) carries information from DNA to the ribosomes - Ribosomal RNA (rRNA) and transfer RNA (tRNA) are involved in protein synthesis - Other RNAs involved in regulating gene expression and processing transport of RNAs and proteins (miRNA, lncRNAs, etc) - RNA can also catalyse some chemical reactions

Answer 13

Nucleotides: ■ 5 - carbon sugar - DNA = deoxyribose - RNA = ribose ■ Nitrogenous base ■ 1 or more phosphate groups linked to the 5’ carbon of the sugar DNA and RNA are polymers of nucleotides

Answer 14

Purines: Adenine and Guanine Pyrimidines: Cytosine, Thymine (DNA only) and Uracil (RNA only) Base pairing: Occurs between pyrimidines and purines DNA A = T G ≡ C RNA A = U G ≡ C

Answer 15

* Attached to the 5’ carbon on the 5-C-sugar * 1 or more phosphates attached Example: ATP (Adenosine triphosphate) the principal form of chemical energy within cells.

Answer 16

■ Nucleotides are not exclusively found in DNA or RNA ■ Can be involved in other biochemical processes and pathways within the cell. ■ e.g. ATP, AMP, dAMP, cAMP

Answer 17

* Polymerisation of nucleotides involves phosphodiester bonds between the 5’ phosphate of one nucleotide and the 3’ hydroxyl of another * Gives polynucleotide chains a sense direction: polynucleotides are always synthesized in the 5’ to 3’ direction * Dehydration synthesis: production of a H20 molecule during the reaction * Oligonucleotides are polymers of only few nucleotides * RNA and DNA are polynucleotides and may contain thousands or millions of nucleotides * RNA mainly single stranded * DNA double stranded

Answer 18

* Double helix * Polynucleotides formed by phosphodiester bonds * Strands run anti-parallel * H-bonds pair the bases on opposite strands, which run antiparallel * Bases are on the inside * Inert sugar-phosphate backbone on outside * Structure stabilized by Hydrogen bonds

Answer 19

* Information in DNA and RNA is always conveyed by the order of the bases * DNA is made up of 2 polynucleotide chains that run anti-parallel to each other * Complementary base pairing allows one strand of DNA (or RNA) to act as a template for synthesis of a complementary strand * Nucleic acids are thus capable of self-replication * The information carried by DNA and RNA directs synthesis of specific proteins, which control most cellular activities * While DNA was first discovered and isolated by Friedrich Miescher in 1869, it would take another 75 years before it was shown to hold the genetic blueprint of life…

Answer 20

Griffith’s experiment (1928): There is a substance (transforming principle) in cell extracts that is capable of transforming a non-pathogenic bacterial strain into a pathogenic form Avery, MacLeod and McCarthy Experiment (1944): Digestion of cell extracts with proteases, carbohydrates and RNAse did not destroy the transforming principle but treatment with DNAse did -> DNA is the transforming principle Hersey-Chase Experiment (1952): Radio-labelled DNA (but not protein) from bacteriophages entered bacterial cells upon infection -> DNA is the genetic material

Answer 21

While DNA had been shown to determine the order of amino acids in a protein sequence, it did not infer/prove that DNA itself directs protein synthesis Experiment by Brenner, Jacob and Meselson determined that RNA and not ribosomes are the intermediate messenger molecule between DNA and proteins – identified messenger RNA (mRNA) In addition to mRNA, two other types of RNA molecules are involved in protein synthesis: transfer RNA (tRNA) and ribosomal RNA (rRNA)

Answer 22

64 different codons: 61 specify amino acids 3 are used as stop signals Universal: used by all organisms and Redundant: multiple codons for most amino acids

Answer 23

The order of the bases (A, T, G and C) must specify the genetic information but how exactly does this determine protein structure? 1957 Sickle-cell anaemia studies: First link between genetic variation and an alteration to the amino acid sequence (Glutamic acid (charged)->Valine (hydrophobic)) Patients with this inherited disease had altered haemoglobin molecules resulting from a single amino acid substitution. Indicates that possibly some variation in the DNA sequence correlated to a change in the amino acid sequence (GAG- >GTG) i.e. order of DNA nucleotides determines the order of amino acids ■ With few exceptions (some viruses contain RNA instead of DNA), all organisms utilize the same genetic code—strong support for the conclusion that all present-day cells evolved from a common ancestor. ■ The mode of replication of viral RNA was determined by studies of RNA bacteriophages of E. coli. ■ These viruses encode an enzyme that catalyzes synthesis of RNA from an RNA template (RNA-directed RNA synthesis). ■ Most animal viruses replicate in this way, but one group (RNA tumor viruses) requires DNA synthesis in infected cells. ■ These viruses (now called retroviruses) replicate via synthesis of a DNA intermediate, a DNA provirus. ■ Reverse transcriptase can be used experimentally to generate DNA copies of any RNA molecule. ■ This has allowed mRNAs of eukaryotic cells to be studied using the molecular approaches currently applied to the manipulation of DNA.

Answer 24

■ This hypothesis was initially met with disbelief because it reverses the central dogma. ■ Later, an enzyme that catalyzes synthesis of DNA from an RNA template (reverse transcription) was discovered (reverse transcriptase enzyme). ■ Reverse transcription has other broad implications. ■ It also occurs in cells and is frequently responsible for transposition of DNA from one chromosomal location to another

Answer 25

* In bacteria, most of the DNA encodes proteins (little non-coding DNA), often arranged in operons (groups of genes with related function expressed together) * The E.coli genome is twice the size of H. influenzae: contains 4.6 Mb and 4,000 genes (90% of DNA is protein-coding) * Simplest eukaryotic genome is Saccharomyces cerevisiae (yeast): contains 12 Mb and 6,000 genes (70% of DNA is protein-coding; only 4% of its genes have introns)

Answer 26

Number of protein-coding genes: 21,000 (1.2 % of genome) Very unexpected result (Estimated 100,000) 40% of human proteins are related to proteins in simpler eukaryotes; most involved in basic cellular processes Most unique proteins have similar domains to other species but arranged in different combinations Mice, rats, and humans have 90% of their genes in common Humans and chimpanzees share ~ 98.8% DNA similarity

Answer 27

■ HARs represent conserved genomic loci with elevated divergence in humans. ■ ~ 3,000 of human accelerated regions: enriched in genes related to DNA interaction, transcriptional regulation and neuronal development. ■ If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. ■ NPAS3 (neuronal PAS domain-containing protein) gene contains 14 HARs. ■ NPAS3: Brain-enriched transcription factor (Maria V. Suntsova & Anton A. Buzdin, BMC Genomics, 2020)

Answer 28

* Found in most, but not all eukaryotic genes (not found in histone genes) * Rarely found in prokaryotic genes (90% of DNA is protein-coding) * Found in both plants and animals * Present in low numbers in yeast (70% of DNA is protein-coding) * Intermediate number in C. elegans, Drosophila and Arabidopsis Introns and Exons Similar observations soon made on cloned eukaryotic genes Sequencing of cloned DNAs and cDNAs (complementary DNAs) indicated that the coding region of the mouse β-globin gene is interrupted by two introns that are removed from the mRNA by splicing

Answer 29

Average human gene has 56,000 base pairs (56 kilobases, kb); contains about 10 exons with 4.3 kilobases-kb Introns make up >90% of the average human gene Additional 3’ or 5’ untranslated regions (UTRs) within the exons

Answer 30

Many introns encode functional (non-coding) RNAs and a very small amount encode proteins (nested genes) Others contain regulatory sequences that control transcription and mRNA processing - regulatory sequences very important for expression of different genes in different cell types in a complex organism. Allow exons of a gene to be joined in different combinations – alternative splicing; this gives rise to different proteins from the same gene Alternative splicing is common in complex organisms Human genes: Alternative splicing can yield between 2 and several thousand different mRNAs per gene; Average 6 alternatively spliced mRNAs; Average 4 different proteins per gene

Answer 31

* Investigation of function in the human genome * Non-coding regions don’t encode protein; but they are not ‘junk DNA’ * Roles in: - gene regulation - structure and replication of eukaryotic chromosomes - evolution of eukaryotic genome * Undiscovered roles? Hot bed of research – are there keys to disease mechanisms and novel therapies in this DNA (eg. MiRavirsen for Hep C)

Answer 32

ENCODE project (2003-present) Discovered that 75% of the human genome is transcribed This cannot all be accounted for by protein-coding genes Includes: * Some non-coding RNA molecules produced (tRNAs, rRNAs, long noncoding RNAs and others) * Pseudogenes !!! Many of the genetic alterations responsible for inherited diseases may be due to mutations in non-coding regions, rather than in the protein-coding region itself !!!

Answer 33

Highly repeated DNA sequences account for a large proportion of complex eukaryotic genomes (100s-1000s of copies per cell) Include: Simple-sequence repeats and LINEs or SINEs

Answer 34

Up to thousands of copies (repeats) of short sequences (1-500 nucleotides) arranged in tandem along a section of the chromosome Can be separated on a density gradient based on their GC:AT ratio Repeat sequence DNAs band as “satellites”, separate from main DNA band (satellite DNAs) Simple-sequence repeats are not transcribed (40% of Drosophila genome, 10% of human genome) Role in chromosome structure

Answer 35

Other repetitive DNA sequences are scattered (interspersed) throughout the genome as opposed to being arranged in tandem repeats on a chromosome Their transpositions into random sites, introducing mutations; expected to be harmful to the cell (eg. Cystic fibrosis, muscular dystrophy, inherited cancers) Short-interspersed elements (SINEs): 100-300 bp; 13% of genome; transcribed; unknown function Long-interspersed elements (LINEs): 4-6 kb; 21% of the genome; transcribed; some encode proteins (e.g. reverse transcriptase) but unknown function Both SINEs and LINEs are examples of transposable elements (capable of moving to different sites in genomic DNA) SINEs and LINEs are retrotransposons as their movement to other parts of the genome involves reverse transcription (RNA ® DNA)

Answer 36

Retrotransposons (e.g. LINEs and SINEs) account for at least 34% of human genome *Function/Effect on the Genome:* Depends on their new integration location: - May disrupt the expression of a gene and lead to some inheritable disorders (cystic fibrosis, hemophilia etc.) or cause some cancers - Contributes to genetic diversity as can regulate the expression of nearby genes following transposition

Answer 37

Retrovirus-like elements also move within the genome by reverse transcription; 2-10kb; 8% of human genome DNA transposons move through the genome by being copied and reinserted as DNA sequences; no reverse transcription; 80-3,000 bp; 3% of human DNA

Answer 38

Genomes can contain multiple copies of many genes (some are non-functional). Why? May be required to produce large amounts of certain proteins or RNAs (e.g. histones, rRNAs) Members of a group of related genes (a gene family) with altered functionality and may be transcribed in different tissues or at different stages of development Other members will have undergone mutations that result in loss of function; non-functional gene copies called pseudogenes -> evolutionary relics; increase genome size (~11,000 in humans) Example: Hemoglobin – α and β subunits are both encoded by gene families in the genome, with different members being expressed in embryonic, fetal and adult tissues Gene duplication can arise by: 1. Duplication of a segment of DNA resulting in the transfer of a block of DNA to a new location in the genome, or 2. Duplication by reverse transcription of an mRNA, followed by integration of the cDNA copy into a new site on a chromosome (retrotransposition) – similar mechanism to that used in transfer of repetitive elements; this can lead to an inactive gene copy (processed pseudogene) with no introns or chromosomal sequences that direct transcription

Answer 39

■ Autosomes: Any chromosome that is not a sex chromosome (X or Y chromosome). Humans have 22 pairs of autosomes. ■ Most eukaryotic cells (somatic cells) are diploidand have two copies of each autosomal chromosome and two sex chromosomes. ■ Sex cells (gametes) are haploid (one copy of each chromosome and one sex chromosome.

Answer 40

Chromosomes were proposed as carriers of genes Most cells of higher plants and animals are diploid (2n) Meiosis gives rise to haploid cells (n) Fertilisation gives a diploid organism (2n) Experiments carried out with Drosophila melanogaster demonstrated that while some traits are independent of each, other traits are inherited together (genetic linkage).

Answer 41

A photograph of the complete diploid set of chromosomes groups in homologouspairs and arranged in order of decreasing size

Answer 42

Different staining techniques can be used to distinguish chromosomes and have been used to map chromosome regions and genes. G-banding: By staining with Giemsa heterochromatin (AT-rich) stains darkly forming G-bands, while less densely packed euchromatin (GCrich) stains more lightly forming R bands. Q-banding: Quinacrine banding. C-banding: constitutive heterochromatin.

Answer 43

* As cells enter mitosis, chromosomes become highly condensed for distribution to the daughter cells * Loops within interphase chromatin fold upon themselves and condense 10,000 fold to highly compact metaphase chromosomes * No longer transcriptionally active * Exact folding has not been defined but it appears to be highly reproducible

Answer 44

Centromere: Specialised region of the chromosome that plays an integral role in ensuring the correct distribution of duplicated chromosomes to daughter cells during mitosis Interphase: DNA is replicated – 2 copies of each chromosome (humans: 46 chromosomes become 46 pairs of sister chromatids)

Answer 45

Centromere is a constricted chromosomal region: - Site where sister chromatids are joined - Attachment site for microtubules of the mitotic spindle fibers Kinetochore: Specialized structure formed by proteins at the centromere; Proteins form attachment between chromatid and microtubule, and also act as motors to move chromatid along spindle fibers to poles of cell Aside from yeast, specific eukaryotic DNA sequences that mediate centromere function have not been identified. Centromeres do have a unique chromatin structure In centromeres: - Histone H3 is replaced by a H3-like variant (CENPA/Centromere Protein A) - CENP-A is found in all studied eukaryotic centromeres - CENP-A containing nucleosomes are required to assemble other kinetochore proteins

Answer 46

DNA sequences of centromere regions were first defined in yeast Followed the segregation of plasmids during mitosis * Functional centromeres: equal plasmid distribution among daughter cells * Non-functional centromeres: many daughter cells fail to inherit plasmid DNA Identified sequences important for centromere function

Answer 47

Unique chromatin structure allows centromeres to be stably maintained at cell division Example of epigenetic inheritance Epigenetic inheritance is the transfer of information to progeny that is not based on DNA sequences. The information is carried on the histones When chromosomal DNA replicates, the parental nucleosomes are distributed to the 2 progeny strands These CENP-A-containing nucleosomes direct the assembly of new CENP-A-containing nucleosomes into chromatin

Answer 48

Telomeres are the sequences at the ends of eukaryotic chromosomes Critical roles in chromosome replication and maintenance Identified after it was noted that broken chromosomes were highly unstable in eukaryotic cells – must be some specific sequences needed at the ends (termini) of chromosomes to confer stability *Tetrahymena (protozoan)* -Telomeres from Tetrahymena were added to the ends of linearised yeast plasmid DNA -Plasmids gained the ability to replicate in a linear form -Conclusion: Telomeric DNA sequences are required for the replication of linear DNA molecules -DNA sequences of telomeres from various eukaryotes are similar -Repeats of simple-sequence DNA; clusters of G residues on one strand -Repeated 100s-1000s of times Telomere maintenance may be important in determining the lifespan and reproductive capacity of the cell

Answer 49

The ends of linear chromosomes cannot be replicated by DNA polymerase DNA polymerase extends a growing DNA chain (Lecture 5) but it cannot start replicating from the end (terminus) of a linear DNA molecule Telomerase plays a critical role in replicating the ends of linear DNA molecules Telomerase: Reverse transcriptase enzyme that replicates telomeric DNA sequences Because of their importance in reproductive capacity and cellular lifespan, telomeres and telomerase are the subject of many aging and cancer studies

Answer 50

Chromatin: complexes between eukaryotic DNA and proteins Experiments using nuclease (an enzyme that breaks down DNA (DNAse)) to digest chromatin yielded nucleosome core particles (beads on electron microscopy): *Nucleosome:* - 147 base pairs; 1.67 turns around a histone core - Histone core: 2 molecules of H2A, H2B, H3 and H4 (core histones) -Basic structural unit of chromatin -DNA wrapped around a histone core -Described by Roger Kornberg (1974) *Chromatosome:* - 166 base pairs; 2 full turns around the histone core - held in place by one molecule of histone H1 (linker histone) * Chromatin condensation has an important role in regulating gene expression * Two forms of chromatin: Euchromatin and Heterochromatin * Euchromatin: chromatin is less condensed and distributed throughout the nucleus; most interphase chromatin exists in this form (cells are not dividing); transcription is active; DNA can be replicated ahead of cell division * Heterochromatin: chromatin is in a highly condensed state; 10 % of interphase chromatin is heterochromatin; transcriptionally inactive; can contain highly repeated DNA sequences or genes that are not expressed in those specific cells

Answer 51

Major proteins of chromatin - Small, basic proteins (high arginine/lysine content) - Facilitates binding to negatively charged DNA - Five major types (H1, H2A, H2B, H3, H4) - Highly conserved among eukaryotes

Answer 52

a kind of cytogenetic technique which uses fluorescent probes to bind parts of the chromosome that show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome

Answer 53

■ Looping of DNA to bring long-range regulatory elements close together. ■ Local (cis) gene regulation ■ Cross-chromosome (trans) gene regulation

DNA Flashcards

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