Gene Expression and Regulation

Chapter 15: The Chromosomal Basis of Inheritance - - - - - Dihybrid crosses where both traits are heterozygous produce a 9:3:3:1 ratio of double dominant : single dominant : single dominant : double recessive T.H. Morgan used flies for his experiments because 1. They reproduce quickly (for larger sample size) 2. Have sex chromosomes (unlike Mendel’s pea plants)  Female is XX  Male is XY  For sex-linked recessive traits, male only need one of the alleles on the X chromosome but females need the allele on both X chromosomes Linked Genes: genes located near each other on the same chromosome tend to be inherited together in genetic crosses o Mendel did not encounter these o Morgan’s lab created a double testcross to measure how these traits were passed Parental Types: offspring that are matching the parents (phenotypically) Recombinant Types: offspring that are not matching one of the parents (phenotypically) Genetic Map: ordered list of the genetic loci along a particular chromosome o Recombinant frequency depends on the distance between genes on a chromosome  The farther apart two genes are, the higher the probability that a crossover will occur between them and therefore the higher the recombination frequency o Linkage map: genetic map based on recombination frequencies o Map Units: distance between genes  One map unit = 1% recombination frequency  If it is over 50%, the genes are on different chromosomes Nondisjunction: the members of a pair of homologous chromosomes do not move apart properly during meiosis I or sister chromatids fail to separate during meiosis II o One game receives two of the same type of chromosome and the other gamete receives no copy DNA in mitochondria and cytoplasm are passed down to offpring o Mitochondrial DNA comes only from the mother Unit 6: Gene Expression and Regulation Chapter 16: The Molecular Basis of Inheritance - - - - - - T.H. Morgan discovered that genes exist as parts of chromosomes Watson and Crick!! Griffith tried to develop a vaccine against pneumonia o Two strains: pathogenic and non-pathogenic o When the pathogenic bacteria were killed with heat and mixed with the nonpathogenic strains, some of the living cells became pathogenic  Some chemical component of the dead pathogenic cells caused this heritable change, known as transformation Transformation: a change in genotype and phenotype due to the assimilation of external DNA by a cell Hershey and Chase proved that DNA was genetic material, not proteins o After Griffith’s experiment, scientists were skeptical of what genetic material was because viruses have a protective coat that is often made of protein o Used a radioactive isotope of sulfur to tag proteins in one batch and radioactive isotope of phosphorus to tag DNA in a second batch  Tested the samples shortly after infection and found that the phage DNA entered the host cells but not the phage protein  Cells released phages that contained some radioactive phosphorus  DNA inside the cell played an ongoing role during the infection process Chargaff discovered that ration of A:T and C:G is approximated 1 because the base pairs appear in nearly equal amounts o Base composition (percentage of each base) different among different species  DNA contributes to genetic diversity Double Helix: presence of two strands in DNA Antiparallel: subunits of DNA run in opposite directions Adenine pairs with thymine and cytosine pairs with guanine o Thymine is replaced with uracil in RNA o Purines: adenine and guanine  Two organic rings o Pyrimidines: cytosine and thymine  Single organic ring o Difference in sizes of pyrimidines and purines accounts for the asymmetry in DNA o Weak interactions known as hydrogen bonds Semiconservative Model: when a double helix replicates, each of the two daughter molecules will have one old strand from the parental molecule and one new strand Origins of Replication: short stretches of DNA having a specific sequence of nucleotides o Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication “bubble” o Having hundreds of origins of replications starts multiple replication bubbles that speed up the process Replication Fork: Y-shaped region where the parental strands of DNA are being unwound at the end of reach replication bubble o Helicases: enzymes that untwist the double helix, separating the two parental strands and making them available as template strands - - - - - - o Single Strand Binding Proteins: bind to the unpaired DNA strands, keeping them from re-pairing o Topoisomerase: relieves strain caused by the untwisting of the double helix by breaking, swiveling, and rejoining DNA strands Unwound sections of parental DNA strands are now available to serve as templates for the synthesis of new complementary DNA strands. Enzymes that synthesize DNA cannot initiate the synthesis o Primer: DNA synthesis is actually a short stretch of this RNA strand o Primase: enzyme that synthesis primer  Starts a complementary RNA chain from a single RNA nucleotide, adding more  5-10 nucleotides long  New DNA strand will start from the 3’ end of the RNA primer DNA Polymerases: catalyze the synthesis of new DNA by adding nucleotides to a preexisting chain o Two main ones are DNA polymerase III and DNA polymerase I o Most require a primer and DNA template strand Antiparallel Elongation o DNA polymerases can add nucleotides only to the free 3’ end of a primer or growing DNA strand  New DNA strand only elongations from the 5’  3’ direction  Leading Strand o To elongation the other new strand of DNA in 5’  3’, DNA polymerase III must work along the other template strand in the direction away from the replication fork  Lagging Strand  Synthesized discontinuously as a series of fragments known as Okazaki fragments  DNA ligase joins the Okazaki fragments Summary of DNA Replication: 1. Helicase unwinds the parental double helix 2. Molecules of single-strand binding protein stabilize the unwound template strands 3. The leading strand is synthesized continuously in the 5’ to 3’ direction by DNA polymerase III 4. Primase begins synthesis of the RNA primer for the fifth Okazaki fragment 5. DNA polymerase III is completing synthesis of the 4th fragment and when it reaches the RNA primer on fragment 3, it will detach and begin adding nucleotides to the 3’ end of the fragment 5 primer in the replication fork 6. DNA polymerase I removes the primer from the 5’ end of fragment 2, replacing it with DNA nucleotides adding one by one to the 3’ end of fragment 3. After the last addition, the backbone is left with a free 3’ end. 7. DNA ligase joins the 3’ end of fragment 2 to the 5’ end of fragment 1 Mismatch Pair: other enzymes remove and replace incorrectly paired nucleotides that have resulted form replication errors Nuclease: DNA-cutting enzyme o When there is an error in replication, the incorrect part is cut out by nuclease and replaced with nucleotides using the undamaged strand as a template o Ex: nucleotide excision repair Once a mismatched nucleotide pair is replicated, the sequence change is permanent in the daughter molecule and in any subsequent copies o Mutation: permanent change in DNA  Can change the phenotype of an organism - - Telomeres: eukaryotic chromosomal DNA molecules have these nucleotide sequences at their ends o Do not have genes, but multiple repetitions of one short nucleotide sequence o Two protective functions: 1. Specific proteins associated with telomeric DNA prevent the staggered ends of the daughter molecule from activating the cell’s systems for monitoring DNA damage 2. Telomeric DNA acts as a buffer zone that provides some protection against the organism’s genes shortening (do not prevent the erosion, simply postpone it) o Become shorter during every round of replication o Telomerase: enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells to restore their original length o Cells from large tumors have unusually short telomeres because they have undergone many cell divisions Chromatin: complex of DNA and protein (histones) fits into the nucleus through a multilevel packing system Chapter 17: Gene Expression - - - - - - Gene Expression: process by which DNA directs the synthesis of proteins One Gene – One Enzyme Hypothesis: each gene controls phenotypes by reflecting in the person’s ability to produce a specific enzyme o Beadle and Tatum studied different mutants and identified mutants that could not survive on minimal medium because they were unable to synthesize certain essential molecules  Took samples form the mutant growing on complete medium and distributed them to different vials that each contained minimal medium and a single additional nutrient. The particular supplement that allowed growth indicated the metabolic defect. Bridge between DNA and protein synthesis is RNA o RNA has ribose instead of deoxyribose as its sugar and uracil instead of thymine as one of its nitrogenous bases o RNA is usually single stranded Transcription: synthesis of RNA using information in DNA o DNA strand provides a template to form mRNA Messenger RNA (mRNA): carries a genetic message from the DNA to the protein-synthesizing organelle Translation: synthesis of a polypeptide using the information in the mRNA o Ribosomes: molecular complexes that facilitate the orderly linking of amino acids into polypeptide chains o By reading 3 letter codons In prokaryotes, DNA is not separated by a nuclear membrane from ribosomes o Lack of compartmentalization allows translation of mRNA to begin while its transcription is still in progress Transcription of a protein-coding eukaryotic gene yields pre-mRNA o Primary Transcript: initial RNA transcript from any gene, including those specifying RNA that is not translated into protein Triplet Code: genetic instruction for a polypeptide chain are written in the DNA as a series of threenucleotide words o Codons: mRNA nucleotide triplets  Anticodon is on the tRNA - - - - RNA Polymerase: pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand o Promoter: where the RNA polymerase attaches and initiates transcription  Begins at the start point and continues for several nucleotides  TATA box: a crucial promoter sequence in forming the initiation complex at a eukaryotic promoter o Transcription Unit: stretch of DNA downstream from the promoter that is transcribed into an RNA molecule Transcription Factors: a collection of proteins that mediates the binding of RNA polymerase and the initiation of transcription Transcription Initiation Complex: whole complex of transcription factors and RNA polymerase II bound to the promotor Enzymes add nucleotides to the 3’ end of the growing RNA molecule Transcribed terminator (RNA sequence) functions as a termination sequence that causes the polymerase to detach from the DNA and release the transcript RNA Processing: both ends of the primary transcript are altered o Interior sections of the RNA molecule are cut out and the remaining parts are spliced together  RNA Splicing: removal of large portions of RNA molecule that is initially synthesized  Introns are removed and the exons only remain  Spliceosome: a large complex of proteins and small RNAs that do RNA splicing  Alternative RNA splicing: genes that give rise to multiple polypeptides depending on which segments are treated as exons during splicing  i.e. one piece of mRNA can code for multiple proteins depending on the introns and exons o 5’ Cap: modified form of a guanine nucleotide added onto the 5’ end after transcription o Poly-A Tail: an enzyme adds 50-250 adenine nucleotides at the 3’ end o 3 main functions: 1. Facilitate the export of mature mRNA from the nucleus 2. Protect the mRNA from degradation by hydrolytic enzymes 3. Help ribosomes attach to the 5’ end of the mRNA once it reaches the cytoplasm Transfer RNA (tRNA): transfers amino acids from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome o The anticodon pairs to the codon on the mRNA o Is terminated when the tRNA reaches the stop codon in the mRNA Mutations: changes to the genetic information of a cell that re responsible for diversity of genes found among organisms o Point Mutations: changes in a single nucleotide pair of a gene o Nucleotide-Pair Substitution: replacement of one nucleotide and its partner with another pair of nucleotides  Silent Mutations: has no observable effect on the phenotypes  This happens because multiple different codons may code for the same protein  Missense Mutations: substitutions that change one amino acid to another  Nonsense Mutations: a point mutation that causes the codon to change and code for a stop codon o Insertions and Deletions: additions or losses of nucleotide pairs in a gene  Frameshift Mutation: the number of nucleotides inserted or deleted is not a multiple of three -  Each codon and the respective amino acid is changed Mutagens: physical and chemical agents that interact with DNA in ways that cause mutations Gene: a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule Chapter 27: Bacteria and Archaea - - - Evolution cannot occur without genetic variation o Leads to individuals that are genetically better equipped for their environment and tend to survive and reproduce at higher rates Even though new mutations are rare, they can increase genetic diversity quickly in a species with short generation times and large populations Genetic Recombination: combining of DNA from two sources o Mutations are rare, so genetic diversity is created this way Horizontal Gene Transfer: when individuals of different species move genes from one organism to another Transformation: the genotype and possible phenotype of a prokaryotic cell are altered by the uptake of foreign DNA from its surrounding Transduction: phages carry prokaryotic genes from one host cell to another o Results from accidents that occur during the phase replicative cycle o A virus that carries prokaryotic DNA may not be able to replicate because it lacks some or all of its own genetic material, but it can attach to another prokaryotic cell and inject prokaryotic DNA from the first cell into it  Virus acts as a messenger of DNA between the two prokaryotic cells Conjugation: DNA is transferred between two prokaryotic cells that are temporarily joined o Typically of the same species Bacteria copy and pass on a small circle of DNA called a plasmid R plasmids are worrisome because they can evolve to develop antibiotic resistance and have genes that allow DNA transfer from one cell to another, enabling the mutation to spread Chapter 18: Regulation of Gene Expression - - - Natural selection favors bacterial that express only genes whose products are needed by the cell because they conserve resources and energy Metabolic control occurs on two levels: 1. Cells can adjust the activity of enzymes already present  Feedback inhibition 2. Cells can adjust the production level of certain enzymes  Regulate the expression of the genes encoding the enzymes Operator: switch segment of a DNA o Coordinately Controlled: a single “on-off switch” can control a cluster of functionally related in genes o Located within or near the promoter Operon: entire stretch of DNA required for enzyme production o The operator, promoter, and genes they control o Only in prokaryotes! o Repressor: switches the operon on or off  Binds to the operator and blocks attachment of RNA polymerase to the promoter, preventing transcription of the genes - - - - - - - o Regulatory Gene: makes the repressor proteins  Located some distance from the operon  Has its own promoter o Corepressor: a small molecule that cooperates with a repressor protein to switch an operator off o Repressible Operon: transcription is usually on but can be inhibited when a small specific molecule binds allosterically to a regulatory protein  Repressible Enzymes: synthesize essential end products from raw materials  Suspends production of an end product when there is enough to save energy and resources  Anabolic pathways o Inducible Operon: transcription is usually off but can be stimulated when a specific small molecule interacts with a regulatory protein  Inducer: inactivates the repressor  Inducible Enzymes: synthesis is induced by a chemical signal  Break down a nutrient to simpler molecule  Producing enzymes only when the nutrient is available to save energy and resources  Catabolic pathways General Transcription Factors: factors that are essential for the transcription of all protein-coding genes o Ex: TATA box within the promoter o May bind to proteins, including other transcription factors and RNA polymerase II Specific Transcription Factors: high levels of transcription of particular genes that depend on the interaction of control elements with these factors Cyclic AMP (cAMP): positive gene regulation o Catabolite Activator Protein (CAP): activator protein that binds to DNA and stimulates transcription of a gene o Prokaryotes and eukaryotes! Histone Acetylation: promotes transcription by opening up the chromatic structure o Eukaryotes only! o Addition of methyl groups leads to condensation of chromatin and reduced transcription o DNA Methylation: a different set of enzymes can methylate certain bases in the DNA itself o Individual genes are more heavily methylated in cells in which they are not expressed  Removal of extra methyl groups can turn these genes on o Once methylated, cells stay that way through cell divisions Epigenetic Inheritance: inheritance of traits transmitted by mechanisms not involving the nucleotide sequence itself o Chromatin modifications can be reversed as opposed to mutations microRNAs (miRNAs): small single-stranded RNA molecules capable of binding to complementary sequences in mRNA molecules o Degrades the target mRNA or blocks its translation Differential Gene Expression: the expression of different genes by cells within the same genome o Happens because of alternative DNA splicing o Alternative RNA Splicing: different mRNA molecules are produced from the same primary transcript depending on which RNA segments are treated as exons and introns o All cells of multicellular organisms have the same DNA so this specializes the cells Cytoplasmic Determinants: maternal substances in the egg that influence the course of early development o Unevenly distributed throughout the cell so the daughter cells are different after meiotic divisions  leads to genetic diversity o Homeotic Genes: control pattern formation in the late embryo, larva, and adult  Patter Formation: cytoplasmic determinants and inductive signals contribute to the development of spatial organization in which the tissues and organs of an organism are all in their characteristic places Chapter 21: Genomes and their Evolution - - The number of genes greatly varies between prokaryotes and eukaryotes o Bacteria and archaea have fewer genes o Within eukaryotes, the number of genes in a species is often lower than expected Eukaryotes generally have larger genomes but fewer genes in a given number of base pairs o Lower gene density that prokaryotes In bacterial genomes, most DNA codes for protein, tRNA, or rRNA, o In eukaryotic genomes, most of the DNA codes for neither protein nor is transcribed. The DNA includes more complex regulatory sequences  Large amounts of introns that are removed from the mRNA code  Only 1.5% of the genome codes for protein or RNA Chapter 20: DNA Tools and Biotechnology - - - Restriction Enzymes: enzymes that cut DNA molecules at a limited number of specific locations o Protect the bacterial cell by cutting up foreign DNA from other organisms or phages o Restriction Site: very specific at recognizing a particular short DNA sequence and cutting both DNA strands at a precise location within this site o Use during DNA cloning to join DNA fragments together 1. Restriction enzymes cut the sugar-phosphate backbones at each arrow o Results in sticky ends 2. DNA fragment form another source is added and base pairing of sticky ends produces various combinations  The other DNA molecule is cut by the same restriction enzyme (same sticky end) 3. DNA ligase seals the strands to produce recombinant DNA molecule / plasmid o Sticky End: the single-stranded portion from the resulting double-stranded restriction fragment Gel Electrophoresis: used to separate and visualize DNA fragments of different lengths o Uses a gel made of a polymer as a molecular sieve to separate out a mixture of nucleic acids of proteins  Shorter molecules are slowed down less than longer ones by the “sieve” and travel further Gene Therapy: introduction of genes into an afflicted individual for therapeutic purposes o Great potential to treat disorders that are traceable to a single defective gene Chapter 19: Viruses - Virus: infectious particle consisting of nucleic acid enclosing in a protein coat o Sometimes surrounded by a membranous envelope Discovered near the end of the 19th century Smaller than cells Capsid: protein shell enclosing the viral genome o Rod-shaped, polyhedral, etc. depending on virus Viral Envelopes: surrounds the capsid and contain host cell phospholipids and membrane proteins - - - - - Bacteriophages / Phages Host Range: each particular virus can infect cells of only a limited number of hose species o Results from the evolution of recognition systems from the viruses  “lock-and-key” system between viral surface proteins and specific receptor molecules on the outside of cells  Cell must have the matching receptor molecule for the virus to bind to it Lytic Cycle: phage replicative cycle that culminates in the death of the host cell o Bacterium lyses and releases the phages that were produced within the cell that can then infect healthy cells causing a series of lytic cycles o Virulent Phage: phage that replicates only by lytic cycle o Restriction enzymes restrict the ability of the phage to replicate within the bacterium because it is methylated in a way that prevents attack Lysogenic Cycle: allows replication of the phage genome without destroying the host o Temperate Phages: phages capable of using the lytic and lysogenic cycle o The DNA molecule of the virus is incorporated into a specific site of the cell’s DNA by viral proteins o Prophage: viral DNA integrated into the bacterial chromosome  One prophage gene codes for a protein that prevents transcription of most other prophage genes, so the phage genome remains silent o Prophages are capable of generative active phages that lyse their host cells  i.e. enter lytic cycle because of certain “triggers” o A few prophage genes may be expressed during lysogeny (not the norm though) and alter the phenotype Retrovirus: equipped with reverse transcriptase which transcribes an RNA template into DNA o Reverse Transcriptase: enzyme which converts RNA to DNA o Ex: HIV Provirus: integrated viral DNA o Never leaves the host’s genome Exam Notes Chemistry of Life - - Hydrogen bonds contribute to the 3-D shapes of proteins and nucleic acids Water molecules can form a maximum of four hydrogen bonds at a time Properties of carbon o 4 valence electrons  form up to 4 covalent bonds (single, double, or triple) o Can form larger molecules o Chains, ring-shaped, or branched Isomers: molecules that have the same molecular formula but differ in their arrangement of these atoms Molecular Genetics - Griffith discovered transformation when a heat-killed nonpathogenic and pathogenic strain killed mice when mixed Hershey and Chase proved that genetic material is DNA by radioactively tagging sulfur in proteins to prove that they didn’t infect the virus in bacteriophages Left is 5’ to 3’ and right is 3’ to 5’ Steps of DNA replication - - - - 1. Begins at origin of replication 2. Intiation proteins bind to origin of replication and separate DNA in both directions (replication bubble) 3. DNA polymerases catalyze the elongation of new DNA at the replication form 4. DNA polymerase adds nucleotide to the growing chain in the 5’ to 3’ direction 5. Replication strand (5’ to 3’) is the leading strand. The lagging strand (3’ to 5’) is copied in a series of segments 6. Lagging strand is synthesized in separate pieces (Okazaki fragments) and are sealed together by DNA ligase Nucleases: remove incorrectly placed nucleotides (nucleotide excision repair) and fill the gap with correct nucleotides Telomeres: repetitive caps at the 5’ end of DNA to prevent degradation DNA is highly extended and available for transcription (not tightly bound to histones) during interphase (euchromatin) Heterochromatin: not available for transcription One gene-one polypeptide hypothesis No RNA processing in prokaryotes (RNA splicing removes introns and spliceosomes join exons and catalyzed by smRNA/ribozyme) o Eukaryotes add a 5’ cap and poly-A-tail to prevent degradation Codons are written in 5’ to 3’ direction RNA polymerase can only add to the 3’ direction (elongates from 5’ to 3’ direction) o Attached to promotor and stops at terminator o Assisted by transcription factors Ribosome Sites: o P-Site: holds tRNA that carries the growing polypeptide chain o A-Site: holds tRNA that carries the next amino acid to be added o E-Site: exit site for tRNA Methylation happens in DNA but acetylation happens on the histones Restriction Enzymes: cut strand of DNA at specific locations (restriction sites) o Serve as protection against invading viruses o DNA ligase joins sticky ends from the leftover restriction fragments