Genetics - Exam #2

Intragenic Suppressors

- A suppressor that occurs through mutation elsewhere in the same gene. - For example, if an initial mutation was caused by deletion of two base pairs, an intragenic suppressor could be a compensatory insertion of two base pairs near the site of the initial mutation, restoring the allele to a near wild-type form.

Tandem vs. Dispersed Duplications

- A tandem duplication means that the duplication is adjacent to the original region. - A reverse tandem duplications means that the duplication is adjacent to the original region, but that the order is reversed (BCCB). - A terminal tandem duplication means that the duplication is adjacent to the original region and is at the end of the chromosome. - A dispersed duplication means that the duplication is not adjacent to the original region, and may be in the same order or the reverse order.

Histones

- About one half of the protein content of chromatin is histone protein. - The histones are five small, basic proteins that are positively charged and bind tightly to negatively charged DNA. - The five types are designated H1, H2A, H2B, H3 and H4. - Among eukaryotes, there is a very strong evolutionary conservation of the amino acid sequences of histone proteins.

Chromosomal Rearrangements

- Affect many genes at one time.

rRNA

- Along with numerous proteins, helps form the large and small ribosomal subunits that unite for translation of mRNA. - In prokaryotes, 50S + 30S make up the 70S functional ribosome. - In eukaryotes, 40S + 60S make up the 80S functional ribosome. - rRNA is the most abundant type of rRNA. - In E. coli, all rRNA genes are transcribed in one long piece that is later cut - this assures they will be present in equal ratios.

Aneuploidy & Cancer

- Aneuploidy is known to be an efficient mechanism for altering the properties of cells, and aneuploid cells are found in virtually all solid tumors. - It is unknown whether aneuploidy events cause cancer or are an effect of cancer.

Unbalanced Translocation

- Arises from a chromosome break and subsequent reattachment to a nonhomologous chromosome in a one-way event.

Autopolyploids vs. Allopolyploids

- Auto = all from one species. - Allo = chromosomes from different species. Some allopolyploids have agriculturally desirable traits derived from two species and are created by chromosome doubling in germ cells.

RNA Polymerase - Bacterial

- Bacterial RNA polymerase is composed of a pentameric (five-polypeptide) RNA polymerase core that binds to a sixth polypeptide, called the sigma subunit, which induces a conformational change in the core enzyme that switches it to its active form. - In its active form, the RNA polymerase is described as a holoenzyme, a term meaning an intact complex of multiple subunits with full enzymatic capacity.

DNA Grooves

- Base-pair stacking creates two grooves in the double helix, gaps between the spiraling sugar-phosphate backbones that partially expose the nucleotides. - The alternating grooves are known as the major groove and minor groove. The major groove is approximately 12A wide and the minor groove is approximately 6A wide. - The major and minor grooves are regions where DNA-binding proteins can most easily make direct contact with nucleotides along one or both strands of the double helix.

Pseudolinkage

- Because alternate segregation patterns produce viable progeny, genes near breakpoints act as if linked.

G-Banding

- Begins with the growing of cells in culture followed by the use of trypsin to stop the cycle in or just before metaphase. - Individual cells from the arrested cell culture are then dropped onto a microscope slide. This bursts the cells and ruptures the nuclear membrane, allowing the chromosomes to spill out. - After some additional treatment, any one of several different dyes or stains can be used on the chromosomes to reveal regional differences in chromatin compaction that produce a series of alternating chromosome bands. - Regions in the chromosome that stain lightly tend to be euchromatic, rich with guanine and cytosine and more transcriptionally active whereas regions that stain darkly tend to be heterochromatic, rich with adenine and thymine and less transcriptionally active. - Not well understood - thought to reflect uneven packaging of loops.

Insertion

- Block of one or more DNA pairs is added.

Deletion

- Block of one or more DNA pairs is lost.

tRNA

- Carries amino acids to ribosomes and binds there to mRNA by complementary base pairing in order to deposit the amino acids to elongate the polypeptide. - About 50 tRNAs are known in bacteria. - The minimum is 20 (one for each amino acid) and the maximum is 61 (one for each codon). The difference is due to the fact that some anti-codons can bind to more than one codon. This is called "wobble" and occurs in the third position of the codon.

Heterochromatin

- Chromosome regions in which chromatin is tightly condensed and contain many fewer expressed genes. - More likely to contain repetitive DNA sequences. - Darkly staining chromosome regions of G-banded chromosomes.

Inversion

- 180° rotation of piece of DNA.

2-Aminopurine (2-AP)

- A base analog with two distinct forms. - In one form, 2-AP acts as an analog of adenine that pairs with thymine. This leads to A-T to G-C transition mutations. - In its other form, 2-AP is protonated. In this form it mispairs with cytosine and produces G-C to A-T transition mutations.

5-Bromouracil (5-BU)

- A base analog. - A derivative of uracil and is very similar to thymine in size and shape. In cells it acts as an analog of thymine. - 5-BU exists in three tautomeric forms that have different base pairing properties. The keto form is complementary to adenine and the enol and ion forms are complementary to guanine. - This means that 5-BrU can be present in DNA either opposite adenine or guanine, resulting in a transition mutation.

Silent Mutation

- A base-pair substitution producing an mRNA codon specifying the same amino acid as the wild-type mRNA. - Possible because the genetic code is redundant, having 2 to 6 codons for most amino acids.

Nonsense Mutation

- A base-pair substitution that creates a stop codon in place of a codon specifying an amino acid.

Missense Mutation

- A base-pair substitution that results in an amino acid change to the protein. - Protein function may be altered by a missense mutation. The specific consequence of the protein change depends on what kind of amino acid change takes place and where in the polypeptide chain the change occurs.

Nucleotide Base Excision Repair

- A multistep process that identifies and removes modified bases and then replaces the entire nucleotide. - Initiated by DNA glycosylases, which recognize modified bases such as uracil created by deamination of 5-methylcytosine, and other base modifications. - For example, a DNA glycosylase can remove a modified base to generate an AP site. The enzyme AP endonuclease then removes the remainder of the nucleotide, after which DNA polymerase and DNA ligase fill the nucleotide gap and seal the sugar-phosphate backbone.

Nucleotide Base Analogs

- A nucleotide base analog is a chemical compound that has a structure similar to one of the DNA nucleotide bases and therefore can works its way into DNA, where it pairs with nucleotide bases in the DNA duplex. - DNA polymerases are unable to distinguish nucleotide base analogs from normal nucleotide bases due to their similarity in molecular size and shape. Consequently, base analogs are incorporated into DNA strands during replication. - They are likely to undergo tautomeric shifts and therefore cause transition mutations.

Transversion Mutations

- A purine is replaced by a pyrimidine or vice versa.

Promoter

- A regulatory sequence (or sequences) near the 5' end of a gene (upstream from the start of transcription) that acts as the binding location of RNA polymerase and directs RNA polymerase to the start of transcription. - In prokaryotes, two sequences are found in most promoters: -35 and -10 Pribnow box sequences. - In eukaryotes, the promoters are the -25 TATA or Hogness box and the -5 initiator element.

LOD Scores Analysis

- A score greater than 0 indicates the possibility that two genes are linked. - By convention, only a score of 3.0 or greater is considered strong evidence of linkage. - A negative LOD score means a pedigree is better explained by independent assortment.

Translocation - Down Syndrome

- Familial Down syndrome is the cause of 5-10% of Down syndrome cases. - Occurs when one parent is a carrier of a Robertsonian translocation of chromosome 21 to another autosome, most often chromosome 14. - The translocation-heterozygous parent has a normal diploid genotype produced by a complete copy of chromosome 14, a complete copy of 21 and a 14/21 fusion chromosome. The fusion chromosome has lost the short arms of chromosomes 14 and 21, but these contain no critical genetic information, and so the carriers have a normal phenotype. - Three possible patterns of segregation of the three chromosomes are equally likely following formation of the trivalent complex (normal 21, normal 14 and 14/21 fusion). Six possible gametes are produced by these patterns. - When united with a normal gamete, three of the six possible gamete types result in nonviable zygotes. The other three types produce viable zygotes. Two have normal phenotype (one is a carrier) and one has Down syndrome.

Map Distance & Crossing Over

- Genes that are closer together undergo fewer crossing over events and non-crossover gametes will exceed the number of crossover gametes.

Polytene Chromosomes

- Giant chromosomes common to many two-winged flies. - They begin as normal chromosomes, but occur through repeated rounds of DNA replication without any cell division (endoreplication). - Usually found in the larvae.

Transposable Genetic Elements

- DNA sequences of various lengths and sequence composition that have evolved the ability to move within the genome by an enzyme-driven process known as transposition. - Transposition is a mutational event - one that has a biological basis. - Transposable elements exist in dozens of forms that range in size from 50 bp to more than 10 kb. They vary in copy number from a few copies up to hundreds of thousands of copies. - They typically create mutations by their insertion into wild-type alleles. This renders the wild-type allele nonfunctional by making it unable to make a wild-type gene product - insertional inactivation. - Evolutionarily, transposable elements can increase genome size. Many transposable elements seem to have the sole function of increasing their own copy number. As a consequence, organisms carrying certain transposable elements derive no useful benefit from their presence. Alternatively, some transposable elements contain expressed genes that may benefit the organism.

Z-Form DNA

- Has a left-handed twist that gives the sugar-phosphate backbone a zigzag appearance. - Occurs in the presence of a high concentration of positively charged ions. - Only a tiny portion of total cellular DNA is ever in the Z form and its physiological significance in cells is unknown.

Monoploid Plants

- Have many uses - visualize recessive traits directly, introduction of mutations into individual cells, select for desirable phenotypes, hormone treatment to grow selected cells and can be used to create a homozygous diploid. - In most animals monoploids are nonviable. A notable exceptions are male bees, wasps and ants.

Genetic Mutations

- Heritable changes in base sequences that modify the information content of DNA.

Genetic Maps

- Indicate the order and relative distances of genes along each linkage group. - Map units (genetic distances) are expressed in centimorgans. - 1 cM = 1% recombination = 1 genetic map unit.

Robertsonian Translocation

- Involves the fusion of two nonhomologous chromosomes, resulting in a single, larger chromosome and a reduction in chromosome number. - If two pairs of chromosomes fuse by Robertsonian translocation, the number of chromosomes in a genome is reduced to 2n-2. - This is a frequently observed mechanism by which chromosome number evolves in related organisms. - Carriers of a single Robertsonian translocation have one chromosome fusion. The homologs of the fused chromosomes remain separate chromosomes.

LINES & SINES

- More than 45% of the human genome is composed of transposable DNA. - Among the functional transposable genetic elements in the human genome, LINE (long interspersed nuclear elements) and SINE (short interspersed nuclear elements) families of elements stand out because of their relative abundance (7%) and their ability to cause spontaneous human gene mutations. - LINEs are up to several thousand base pairs in length and have an average length of about 900 bp. Almost one million copies are found in the human genome and they collectively constitute a little more than 20% of the total genome sequence. - SINEs are much shorter and have their sequences truncated at one end of the element, likely because the reverse transcription process used for their transfer terminates before the entire sequence has transposed. They constitute 10% of the human genome.

Translation - Termination

- Occurs when one of the three stop codons (UAG, UGA, UAA) appears in the A site of the ribosome.

Polyploid Plants

- Often increases size and vigor, therefore polyploid plants are often selected for agriculture/horticulture uses. - Triploids are almost always sterile and result from the union of monoploid and diploid gametes. - Tetraploids are often a source for new species and result from the failure of chromosomes to separate into two daughter cells during mitosis of the diploid.

Reciprocal Translocation

- Parts of nonhomologous chromosomes change places.

Transcription - Elongation

- Polymerization occurs in the 5'-to-3' direction. - There is no apparent proofreading. - The end product of transcription is a single-stranded RNA that is complementary and antiparallel to the template DNA strand.

Transcription - Termination

- RNA polymerase hits a region of multiple, short, inverted repeats (uracils). This allows the growing RNA to form base pairs within the chain. - The formation of a loop in the newly synthesized RNA is the signal that terminates transcription. - In prokaryotes, a helper molecule called rho is needed to bind with the RNA polymerase.

Transcription - Initiation

- RNA polymerase requires transcription factors to initiate transcription. - The first transcription factor binds to the promoter. - Then, other transcription factors come and open the downstream DNA and bind to RNA polymerase, activating it. - Another transcription factor uses ATP to phosphorylate RNA polymerase II, releasing it from the complex and allowing it to start transcribing. The site of phosphorylation is a long polypeptide tail that extends from the polymerase molecule.

Adjacent-2 Separation

- Rare because it requires that chromosomes that share homologous centromeres move to the same pole of the cell at anaphase I. This is atypical of the usual pattern at anaphase I, in which homologous chromosomes (that carry homologous centromeres) are separated in the reduction division. - None of the gametes or progeny resulting from adjacent-2 separation are viable.

Pericentric Inversion

- Reorients a chromosome segment that includes the centromere.

Genes - Cis and Trans

- Repulsion (trans linkage) occurs when each parent is a single mutant (or recessive for only one of the two traits). - Coupling (cis linkage) occurs when one parent is wild-type and the other is a double mutant. - With independent assortment, cis or trans alternatives are irrelevant to outcome.

Amino-Acyl tRNA Activation

- Requires an enzyme called amino-acyl synthetase. - It first attaches a molecule of ATP to an amino acid, thus activating it. This ATP is needed to provide energy to form the bond between the amino acid and the appropriate tRNA. - Each synthetase is specific for a given amino acid.

Paracentric Inversion

- Results from the inversion of a chromosome segment on a single arm and does not involve the centromere.

snRNA

- Small nuclear RNA. - Found in eukaryotic nuclei, where multiple snRNAs join with numerous proteins to form spliceosomes that remove introns from precursor mRNA.

Transcription

- The cellular process that synthesizes RNA strands from a DNA template strand.

B-Form DNA

- The most common and stable form of DNA. - Has a right-handed twisting of the sugar-phosphate backbone.

Euploidy & Aneuploidy

- The number of chromosomes in nuclei of normal cells is a multiple of the haploid number (n), the number of a single set of chromosomes. - The number of chromosomes is euploid if it is a whole-number multiple of the haploid number. - If cells contain a number of chromosomes that is not euploid, the chromosome number is aneuploid. Aneuploidy occurs when one or more chromosomes are lost or gained relative to the normal euploid number. - Autosomal aneuploidy is harmful to the organism.

Translation

- The process taking place at ribosomes to synthesize polypeptides. - Complementary base pairing between mRNA codons and tRNA anticodons determines the order of amino acids composing the polypeptide. - The process can be divided into four steps: 1. Amino-acyl tRNA formation 2. Initiation 3. Elongation 4. Termination

Translation - Initiation

- The synthesis of every protein in E. coli begins with the amino acid N-formyl methionine, which is often removed before the protein is functional. - The codon for methionine is AUG. This codon has two complementary tRNAs; only tRNA-fmet is used in the initiation complex. - Initiation begins when the small subunit binds to the Shine-Delgarno sequence on the mRNA.

Aneuploidy in Humans

- Theoretically, there are potentially 24 different kinds of trisomy in humans - one for each autosome, and one each for the X and Y chromosomes - and an equal number of potential monosomies. Yet, only autosomal trisomies of chromosomes 13, 18 and 21, and no autosomal monosomies are seen with any measurable frequency in newborn human infants. Multiple forms of sex-chromosome trisomy are detected with some frequency at birth, however, as is one type of sex-chromosome monosomy. Humans can tolerate X chromosome aneuploidy because X inactivation compensates for the extra dosage. - Other trisomies and monosomies occur at conception, but the resulting zygotes never survive to be born alive. - In the first trimester of pregnancy, about half of all human conceptions spontaneously abort, and more than half of the spontaneously terminated human pregnancies carry abnormalities of chromosome number or chromosome structure. This points to a high frequency (15-25%) of meiotic disjunction in humans.

RNA Polymerase - Eukaryotic

- Three different RNA polymerases transcribe distinct class of RNA coded by eukaryotic genomes. - RNA pol I transcribes three ribosomal RNA genes. - RNA pol II transcribes messenger RNAs and most snRNAs. - RNA pol III transcribes all tRNA genes as well as one snRNA gene and one rRNA gene. - Despite differences in sizes and molecular complexity, the RNA polymerases have a similar overall structure, forming a characteristic shape reminiscent of DNA polymerase with a hand composed of protein fingers to help RNA polymerase grasp DNA and a palm in which polymerization takes place.

Translation - Elongation

- To begin elongation, the correct tRNA, GTP and two proteins (elongation factors) are needed. - Elongation continues using the genetic code. - The enzyme peptidyl transferase transfers the peptide chain from the tRNA in the P site to the amino group of the amino acid attached to the tRNA in the A site. - The tRNA with the growing chain is then translocated to the P site, a step requiring GTP.

Patau Syndrome

- Trisomy 13. - 1 in 15,000 live births. - Results in mental retardation and developmental delay, possible deafness, major organ abnormalities, early death, facial deformities, heart defects, kidney abnormalities, malformed or absent eyes and nose, microcephaly, small mouth and cleft deformities, polydactyly.

Edwards Syndrome

- Trisomy 18. - 1 in 8000 live births, 80% female. - Facial deformities, fingers curled over one another in clenched fists, heart defects, kidney abnormalities, low, small ears, microcephaly, small mouth and cleft deformities, spina bifida, club foot.

Recombinants

- Two categories in low frequency combine the phenotypes of the two original parents (P1). - Recombination occurs during prophase I of meiosis. - There is much less recombination in heterochromatin compared to euchromatin.

Parentals

- Two categories in very high frequency have the same phenotypes as the original parent (P1). - Non-recombinants.

mRNA

- Used to encode the sequence of amino acids in a polypeptide. - May be polycistronic (encoding two or more polypeptides) in bacteria and archaea. - Encodes single polypeptides in nearly all eukaryotes. - Once the mRNA is transcribed, it undergoes post-transcriptional modifications in eukaryotes. - At the 3' end, a poly-A tail of 20-200 nucleotides is added, a 7-methyl guanosine cap is added to the 5' end, and introns are removed and exons are spliced together. - Removal of introns and splicing of exons is done with the spliceosome. This is a complex of several snRNA molecules and more than 70 proteins. - Such modifications are not needed in prokaryotes - translation can begin even before transcription is complete.

Partial Chromosome Deletion

- When a chromosome breaks, both strands of DNA are severed at a location called a chromosome break point. The broken chromosome ends at a break point retain their chromatin structure, and they can adhere to one another, to other broken chromosome ends or to the ends of intact chromosomes. - Any part of a broken chromosome that remains acentric (without a centromere) can be lost during cell division. - Chromosome breakage can result in partial chromosome deletion, by the loss of a portion of a chromosome. The size of the deletion and the specific genes deleted are significant factors in the degree of ensuing phenotypic abnormality. - Homozygosity for deletion is often, but not always lethal while heterozygosity is often detrimental.

Mismatch Repair

- When faced with a base-pair mismatch, repair enzymes must correctly identify the nucleotide to be replaced, and this requires distinguishing between the original DNA strand and the new DNA strand with the mismatched nucleotide. The identification is accomplished by the sensitivity of mismatch repair enzymes to methylation of specific nucleotides in the original DNA strand.

Three Point Crosses: Calculations

1. Identify the parents (largest group) and double recombinants (smallest group). 2. Determine which gene is in the middle. 3. Find all region one and two recombinants. 4. Calculate map distance for each. 5. Calculate interference. 6. Coefficient of coincidence.

Replication - Prokaryotes vs. Eukaryotes

Prokaryotes - Begins at a single, fixed location (oriC) - Proceeds at about 1000 nucleotides per second - done in 40 mins. - Includes a proof-reading function. - Three DNA polymerase enzymes: I, II and III. Eukaryotes - Multiple origins (replicons). - Copies at about 50 base pairs per second, done in about three minutes. - Five polymerase enzymes (gamma for mitochondria).

Chromosome Scaffold

- A filamentous nonhistone protein framework that gives chromosomes their shape. - The shape of the chromosome scaffold is reminiscent of the metaphase chromosome structure, consisting of sister chromatids joined at the centromere. - Chromatin loops containing 20,000 to 100,000 bp are anchored to the chromosome scaffold by other nonhistone proteins at sites called matrix attachment regions.

Forward vs. Reverse Mutation

- A forward mutation changes the wild-type to a different allele. - A reverse mutation causes a novel mutation to revert back to the wild-type.

Luria & Delbruck - Fluctuation Test

- Demonstrated that genetic mutations are random rather than a response to a selective pressure. - In their experiment, Luria and Delbrück inoculated a small number of bacteria into separate culture tubes. After a period of growth, they plated equal volumes of these separate cultures onto agar containing the T1 phage (virus). - The two possibilities tested by the Luria-Delbrück experiment were: (A) If mutations are induced by the media, roughly the same number of mutants are expected to appear on each plate. (B) If mutations arise spontaneously during cell divisions prior to plating, each plate will have a highly variable number of mutants. - The second possibility occurred. There was a high variance in mean number of resistant cells per culture.

Chargaff

- Determined that A is paired with T and G is paired with C.

Induced Mutations/Mutagens

- Induced mutations are mutations produced by interaction between DNA and a physical, chemical or biological agent that generates damage resulting in mutation. - The agents generating mutation-inducing DNA damage are called mutagens.

Pseudodominance

- Occurs when a normally recessive allele is unmasked and expressed in the phenotype because the dominant allele on the homologous chromosome has been deleted. - Used to map genes in deleted chromosome regions by deletion mapping.

Duplications

- Presence of extra copies of some chromosome region.

Direct Repair of UV-Induced Photoproducts - Photoreactive Repair

- Pyrimidine dimers, the common photoproducts of UV-induced DNA damage can be directly repaired by photoreactive repair, a DNA repair mechanism found in bacteria, single-celled eukaryotes, plants and some animals, but not in humans. - Photoreactivation utilizes the enzyme photolyase to breaks the bonds formed during pyrimidine dimerization. - A thymine dimer produced by UV irradiation is bound by photolyase. Visible light energy is absorbed by photolyase and is redirected to break the bonds forming the dimer.

Deamination

- The loss of an amino (NH2) group from a nucleotide base. - Each of the DNA nucleotide bases contains an amino group, but deamination of cytosine is the deamination event most associated with mutation. - When cytosine is deaminated, the amino group is replaced by an oxygen atom, forming the nucleotide base uracil. DNA mismatch repair readily recognizes uracil as an RNA nucleotide base and removes it from DNA. The excised uracil is replaced by cytosine, and wild-type sequence is restored. - A different scenario occurs when deamination takes place on a cytosine that has been methylated. A methylated cytosine has the hydrogen atom at the number 5 carbon replaced with a methyl group. Deamination of 5-methylcytosine creates thymine and generates a base-pair mismatch between the newly formed thymine on one strand and the previously complementary guanine on the other strand. - If mismatch repair enzymes correct the mismatch before the next DNA replication cycle, two outcomes are possible: either (1) the repair will restore the wild-type G-C base pair or (2) the repair will generate an A-T base pair transition substitution. - Alternatively, if mismatch repair does not occur prior to replication, one daughter chromatid will be mutant and one will be wild-type. The guanine-containing strand will be used to produce a daughter duplex with the wild-type G-C sequence, while the thymine-containing strand will be used to synthesize a daughter duplex containing a G-C to A-T base pair.

Auxotrophic Mutant

- Unable to make a required nutrient.

Terminal Deletion

- A chromosome break that detaches one arm of a chromosome leads to a terminal deletion. - The chromosome fragment broken off in terminal deletion contains one of the chromosome ends consisting of a telomere and additional genetic material. Without a centromere, the acentric fragment lacks a kinetochore. It is unable to attach spindle fibers and cannot migrate to a pole of the cell during division. Acentric chromosome fragments are lost during cell division. - Ex: Cri-du-chat - caused by terminal deletion of the p arm of chromosome 5.

Imprinting

- A specialized example of resetting of epigenetic patterns in meiosis. - For the small number of mammalian genes subject to genomic imprinting, both copies of the gene are functional, but just one is expressed. - In mammals, two copies of each autosomal gene are inherited - one copy is on a chromosome inherited from the mother, and the other copy is on the homologous chromosome from the father, and usually both gene copies are expressed. For a small number of genes whose expression is subject to genomic imprinting, however, this pattern does not hold. - Instead, one copy of the gene is actively expressed while the other copy is silent. The expressed gene copy is always inherited from a particular parent (for some genes it is the mother, for others it is the father), and the silent copy is the one inherited from the other parent. - In primordial germ-line cells, the inherited imprinting patterns are first erased and then are reestablished in the sex-specific pattern of the germ line early in gametogenesis. - For example, in the female germ line, methylation of the paternal chromosome is reversed by demethylase activity, and the insulator protein is removed from the ICR on the maternal chromosome. Both chromosomes are then re-printed with the female-specific pattern.

Intergenic Suppressors

- A suppressor that is produced by a mutation in a different gene. - For example, an original mutation inactivates gene A and results in the loss of function of the major pigment-transporting protein in a flower. A minor pigment-transporting gene B remains active, transporting a small amount of blue pigment from gene C. The initial mutation produces a light-blue flower. The intergenic suppressor is a mutation of gene B that increases gene transcription and thus increases production of the pigment-transporting protein. The mutation of gene B compensates for the mutation of gene A and restores the wild-type dark-blue flower phenotype.

Alkylating Agents

- Add bulky side groups such as methyl and ethyl groups to nucleotide bases. These added groups are known as bulky adducts. - EMS is a powerful alkylating agent that adds an ethyl group to thymine or an ethyl group to guanine. This and other bulky adducts interfere with normal DNA base pairing and may distort the DNA double helix.

Radiation-Induced DNA Damage

- All forms of radiant agent less than 380nm can cause DNA damage and are mutagenic, including UV radiation, X-rays, gamma rays and cosmic energy. - X-rays and radioactive materials can cause DNA damage in multiple ways, including the induction of DNA single-strand or double-strand breaks. These breaks potentially block DNA replication and thus pose a significant threat to the integrity and survival of affected cells.

Alternate Segregation

- At anaphase I, the normal chromosomes move to one cell pole and the translocated chromosomes move to the opposite the pole. - At the completion of meiosis, all gametes are viable because each contains a complete set of genetic information for the two chromosomes. - Fertilization of a gamete containing the two normal chromosomes will produce a normal zygote, while fertilization of a gamete containing the translocated chromosomes will produce a zygote with reciprocal balanced translocation heterozygosity. - Only this method of segregation produces viable gametes and viable progeny. This pattern accounts for one half of all meiotic events in these individuals - semisterility.

Meselson-Stahl Experiment - Results

- At the time there were three competing models of DNA replication: 1. Semiconservative - each daughter duplex contains one original parental strand of DNA and one complementary, newly synthesized daughter strand. The first cycle would yield two DNA molecules with N15/N14 structure, and the second would yield equal amounts of N14/N14 and N15/N14 structures. 2. Conservative - one daughter duplex contains the two strands of the parental molecule and the other contains two newly synthesized daughter strands. The first cycle would yield one N14/N14 structure and one N15/N15 structure. The second cycle would yield three N14/N14 structures and one N15/N15 structure. 3. Dispersive - each daughter duplex is a composite of interspersed parental duplex segments and daughter duplex segments. This predicts a single DNA density in all generations - a mix of N14 and N15. Semiconservative was the correct mechanism.

300-nm Fiber

- Beyond the 30-nm stage, chromatin compaction and the presence of nonhistone proteins are integral to the structure of chromosomes and the process of chromosome condensation that initiates with the onset of prophase in the M phase of the cell cycle. - Interphase chromosome structure results from the formation of looped domains of chromatin similar to supercoiled bacterial DNA. The loops are variable in size, containing from tens to hundreds of kilobase pairs and consisting of 30-nm-fiber DNA looped on a category of nonhistone proteins that are the foundation of chromosome shape. - The diameter of looped chromatin is approximately 300 nm, so looped chromatin is called the 300-nm fiber. - With continued condensation, the chromatin loops form the sister chromatids. - In metaphase, chromosome condensation reaches it zenith.

Reciprocal Balanced Translocation

- Breaks occur on two nonhomologous chromosomes and the resulting fragments switch places when they are reattached. - One member of each homologous pair is altered and none of the four chromosomes has a fully homologous partner. The absence of complete homology requires formation of a tetravalent synaptic structure to enable homologous regions to synapse during metaphase I. - Two patterns of chromosome segregation emerge from the tetravalent structures found in translocation heterozygotes. Alternate segregation and adjacent-1 segregation each occur in approximately 50% of meiotic divisions. Occasionally, an unusual pattern of segregation known as adjacent-2 segregation takes place.

DNA Intercalating Agents

- Certain small molecular compounds called DNA intercalating agents can squeeze their way between DNA base pairs and distort the duplex. - These compounds can also attach to nucleotide bases to form bulky adducts that contribute to DNA distortion. These helical distortions lead to DNA strand nicking that is not efficiently repaired. - Strand nicking appears to be caused by the intercalating agent interfering with the action of topoisomerase II that is active in relieving DNA supercoiling. In the following replication cycle, the nicked strands can gain or lose one or more nucleotides - can cause frameshift mutations.

Inversion Loop

- Chromosome inversion does, however, cause a difference in linear order of genes between the homologs; thus, to bring the homologs of an inversion heterozygote into synaptic alignment during meiosis requires the formation of an unusual inversion loop at synapsis. However, an organism that is homozygous for an inversion carries the same order of genes and chromosome regions on both homologs and therefore will experience normal chromosome synapsis without the need for inversion loop formation. - Crossing over takes place between the homologs, but whereas crossing over that occurs outside the region spanned by the inversion loop takes place in the normal manner, crossing over inside the region of the inversion loop results in duplications and deletions among the recombinant chromosomes. - Crossover results in two viable gametes and two non-viable gametes.

Chromosome Translocation

- Chromosome translocation takes place following chromosome breakage and the reattachment of a broken segment to a nonhomologous chromosome. - If no critical genes are severed or have their regulation disrupted by the breakage or translocation events, translocation heterozygotes, with one normal chromosome and one altered chromosome in each homologous pair, have a normal outward phenotype and a normal pattern of gene expression. Even if no phenotypic abnormalities are detected, however, certain translocation heterozygotes can experience semisterility as a result of abnormalities of chromosome segregation.

C-Banding

- Chromosomes are treated with acid and base (to denature DNA), then stained with Giesma stain. - Only heterochromatic regions close to the centromeres and rich in satellite DNA stain, except for the Y chromosome - its long arm usually stains throughout.

Tautomeric Shifts

- DNA nucleotide bases are organic chemical structures that can occasionally convert, in what are called tautomeric shifts, to alternative structures known as tautomers. - Tautomers are structures that have the same composition and general arrangement but a slight difference in bonding and placement of a hydrogen. The generation of a tautomer changes the 3D structure of the nucleotide base from a more stable common form to a rare, less stable form. - Tautomeric shifts affecting nitrogenous bases can lead to base-pair mismatch between a rare tautomer on one DNA strand and a common form on the complementary strand. - Mispairing of DNA nucleotides due to the presence of tautomers is the most common form of DNA replication error. - Purine-pyrimidine pairing is maintained, but the rare tautomers form the wrong number of hydrogen bonds, leading to the mispairing with the common tautomeric form of the incorrect nucleotide of the opposite type. - For example, the rare tautomer of the pyrimidine thymine forms three hydrogen bonds instead of the normal two hydrogen bonds, and thus it mispairs with guanine, the wrong purine.

DNA Proofreading

- DNA polymerases are generally able to undertake DNA proofreading to be sure replication is accurate. - DNA polymerases have the ability to synthesize DNA (5'-to-3' polymerase activity) but also to proofread newly synthesized DNA for accuracy and remove erroneous nucleotides. This proofreading ability resides in the 3'-to-5' exonuclease activity of DNA polymerases capable of removing some of the newly laid daughter strand sequence. - Polymerases like pol III and pol I have a structure somewhat like an open hand: a thumb and fingers hold the template and daughter strands in the palm, where 5'-to-3' polymerase activity is centered. When a replication error occurs, the mismatched DNA bases of the template and daughter strands are unable to hydrogen bond properly. As a result, the 3'-OH end of the daughter strand becomes displaced, blocking the further addition of nucleotides and inducing rotation of the daughter strand into the 3'-to-5' exonuclease site at the heel of the hand. - Several nucleotides, including the mismatched one are then removed from the 3' end of the daughter strand, after which the daughter strand rotates back to the polymerase site in the palm and replication resumes.

DNA Replication Initiation

- DNA replication is most often bidirectional, progressing in both directions from a single origin of replication in bacterial chromosomes and from multiple origins of replication in eukaryotic chromosomes. - Once replication is underway, there is expansion around the origin of replication, forming a replication bubble. - DNA replication in E. coli requires that replication-initiating enzymes locate and bind to the consensus sequences at the origin of replication. The first to bind is DnaA, an initiator protein. - A helicase protein then uses ATP energy to hydrolyze bonds joining complementary nucleotides. This hydrolysis separates the DNA strands and unwinds the double helix. The unwound strands of DNA would seek maximum stability by reannealing, re-forming complementary double-stranded DNA, except for the presence of single-stranded binding protein (SSB). Single-stranded binding protein prevents reannealing of the separated strands, keeping them available to serve as templates for new DNA synthesis. - The DNA polymerase enzymes that are responsible for synthesizing DNA strands use the template strand to direct the addition of nucleotides to daughter strands in a complementary and antiparallel manner. These new nucleotides are added to the 3' end of the growing daughter strand, and the overall direction of daughter strand elongation is 5' to 3'. - However, DNA polymerases are unable to initiate DNA strand synthesis on their own. They require the presence of a primer sequence, a short single-stranded segment that begins a daughter strand and provides a 3'-OH end to which a new DNA nucleotide can be added by DNA polymerase. DNA replication is thus initiated by a specialized RNA polymerase, called primase that synthesizes a short RNA primer. - During DNA replication, all DNA molecules undergo some level of superhelical twisting that imparts torsional twisting to the molecule beyond that of the spiraling double helix. Circular chromosomes in particular are closed by covalent bonds (phosphodiester bonds) - superhelical twisting that accompanies DNA replication creates torsional stress that would shear the molecule if it were left uncontrolled. As replication progresses, unwinding of the double helix causes superhelical twisting to accumulate, producing supercoiled DNA. To avoid random breakage in the molecule that could lead to a breakdown of DNA replication, enzymes known as topoisomerases (DNA gyrases) catalyze a controlled cleavage and rejoining of DNA to allow over-wound DNA strands to unwind. Relief of supercoiling is accomplished by cutting either one or both strands of DNA allowing DNA to unwind and then resealing the strands.

DNA Replication - Elongation

- Daughter DNA strands are synthesized at the replication fork by the DNA polymerase III (pol III) holoenzyme, the principal DNA-synthesizing enzyme. - Pol III begins at the 3'-OH end of an RNA primer and rapidly synthesizes new DNA with a sequence complementary to the template strand nucleotides. - Most of the enzymes involved in DNA replication are part of a single large protein complex at each replication fork called the replisome. There is one replisome at each replication fork and each contains, among other components, two complete pol III. In each replisome, one pol III carries out the 5'-to-3' synthesis of one daughter strand continuously, in the same direction in which the replication fork progresses. The second pol III enzyme carries out synthesis of the other daughter strand. The continuously elongated daughter strand is called the leading strand. - The other daughter strand has a 5'-to-3' direction of elongation that runs opposite to the direction of movement of the replication fork. These daughter strands are elongated discontinuously in short segments, each of which is initiated by an RNA primer - called the lagging strand. - Okazaki fragments are the result of discontinuous synthesis of DNA on the lagging strand.

Avery, McCarty & MacLeod Experiment

- Dawson and Avery developed an in vitro transformation procedure to mix living R cells with a purified extract of cellular material derived from heat-killed SIII cells containing the transformation factor. Biochemical assays indicated that the SIII extract consisted mostly of DNA, along with a small amount of RNA and trace amounts of proteins, lipids and polysaccharides. - The Avery, McCarty, McLeod experiment identified the role of DNA in transformation by eliminating lipids, polysaccharides, protein, RNA and DNA one at a time from the SIII extract. - In each experimental trial, the SIII extract was treated to remove one component at a time, and the treated extract was mixed with RII cells. The in vitro transformation reaction was allowed to take place, and the occurrence or prevention of transformation was assessed. - In vitro transformation takes place in the control experiment and when lipids and polysaccharides, proteins or RNA are moved from the extract. The experiment that used DNAse to specifically degrade DNA did not result in transformation. - Conclusion: DNA is the transformation factor and the probably hereditary material.

Euchromatin

- During interphase, chromosome regions containing genes that are actively expressed and have a lesser degree of chromatin condensation. - Lightly staining regions of G-banded chromosomes.

Deletion Mapping Example

- Ex: deletion mapping using pseudodominance to map the Notch gene in Drosophila. - The Notch gene resides on the X chromosome, and its location is revealed by the detection of pseudodominance in fruit flies that are heterozygous for partial X-chromosome deletions. - Pseudodominance appears in females that are heterozygous for the partial deletion, carry the recessive allele on the intact X chromosome and have lost the dominant allele from the other, partial deletion of the X chromosome. - In the example, six different mutants have been deleted. - The first two partial deletions do not lead to pseudodominance, indicating that the regions deleted do not contain the Notch gene. The other two partial deletions result in pseudodominance, indicating that the Notch gene locus containing the dominant allele is in that region. - To home in on the location of Notch, progressively smaller partial deletions are used to identify the smallest deletion segment common to all deletions resulting in pseudodominance. This is where the gene resides.

Imprinting - Huntington's Disease (Juvenile)

- HD (gene) encodes the huntingtin protein that is expressed in brain cells and in other cells of the body. - In mutant form, huntingtin appears to aggregate with itself and other proteins, hastening the death of neurons in the brain that lead to the motor abnormalities - progressive loss of motor control by unintentional and uncontrollable movement - that are characteristic of the disease. - HTD (disease) is caused by increases in the length of gene sections containing end-to-end repeats of three nucleotides (CAG). - Wild-type HD genes vary in the number of CAG repeats, ranging from 6 to 28 repeats in the general population. HD alleles with 28 to 35 CAG repeats do not cause disease, but as a consequence of the increased CAG number, the alleles are unstable and prone to further expansion. - Alleles that have 36 to 40 CAG repeats have expanded beyond the normal range, and the huntingtin protein produced by these alleles can behave abnormally and can result in disease symptoms that show reduced penetrance. Individuals who carry 36 to 40 CAG repeats might or might not develop HD. If they do, disease symptoms have a late age of onset and progress slowly. - Individuals with HD alleles containing more than 40 CAG repeats have HD that can develop at any time from the late teens onward. - The CAG repeat size of the mutant human HD gene is different in male and female progeny from identical fathers. Males predominantly expand the repeat whereas females predominantly contract the repeat. Offspring of affected mothers are more likely to show no change or to show contractions in CAG repeat size, even if the maternal allele is of a similar size to one that expands through paternal transmission.

Nucleosome

- Histones are the principal agents in chromatin packaging and the fundamental unit of histone protein organization is the nucleosome core particle. - The nucleosome core particle is a heterooctameric protein complex that contains two molecules each of four histones - H2A, H2B, H3 and H4. - Nucleosome core particles are flat-ended structures. Each nucleosome core particle is wrapped by approximately 146 base pairs of DNA that twist one and two-thirds turns around the core particle. This wrapping is the first level of DNA condensation, and it condenses the DNA approximately sevenfold. - The 146 bp of DNA wrapped around a nucleosome core particle is called core DNA, and the combination of a nucleosome core particle wrapped with core DNA is identified as a nucleosome. - Electron micrographs of chromatin fibers in a highly decondensed state show a regular series of circular structures strung together by connecting filaments - beads on a string. The beads are nucleosomes that are a little more than 11nm in diameter, and the string is linker DNA - the DNA between regions of core DNA. - If the 146 bp in length of core DNA is added to the length of linker DNA, the nucleosome repeat distance is approximately 160 bp. - This beads-on-a-string form of chromatin is identified as the 10-nm fiber, since the diameter of nucleosomes is approximately 10nm.

Hydroxylating Agents

- Hydroxylation is the addition of a hydroxyl group to a recipient compound by a donor called a hydroxylating agent. - Hydroxylamine is a hydroxylating agent that adds a hydroxyl group to cytosine by displacing an H2, thus creating hydroxylaminoacytosine. This often pairs with guanine, but frequently mispairs with adenine, leading to C-G to T-A base-pair transition mutations.

Nucleotide Excision Repair - E. coli

- In E. coli, NER corrects damaged-induced distortions of the DNA helix. - Proteins required are UvrA, UvrB, UvrC and UvrD. - A complex of two UvrA and one UvrB proteins slides along the DNA. When it encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the UvrB at the lesion. - When UvrBC forms, the UvrC cuts about four nucleotides from the lesion on the 3' side, and about seven nucleotides away on the 5' side. Then UvrB is released and UvrD binds the 5' cut end. - UvrD (a helicase) binds and unwinds the region between cuts, releasing the damaged segment. DNA polymerase I fills the gap. - DNA ligase joins the DNA segments together, and repair is complete.

Mismatch Repair - E. Coli

- In E. coli, methylation is particularly common on the adenine of 5'-GATC-3' sequences. GATC sequences are palindromes, meaning that both strands of DNA will contain a 5'-GATC-3' sequence. - The addition of methyl groups is delayed after the completion of replication, meaning that immediately after replication is complete, original strands contain methylated adenines in GATC sequences, but daughter strands do not. - Two E. coli genes - mutS and mutL - produce enzymes that recognize and bind to DNA containing base-pair mismatches. MutS protein binds first to the mismatch region, and then it recruits MutL protein to the site as well. This begins a series of enzyme-driven steps that recruits a third protein, MutH that breaks a phosphodiester bond on the 5' side of the guanine of a GATC sequence on the unmethylated daughter strand. - Broken phosphodiester bonds are known as "nicks", and the nick generated by MutH attracts an exonuclease that digests a short stretch of nucleotides extending beyond the GATC and including the mismatched base. - DNA polymerase then uses the original strand as a template to fill the gap created by nuclease digestion, and DNA ligase completes the repair. - A specialized methylase enzyme known as Dam methylase then methylates the adenine of the GATC sequence on the daughter strand.

Adjacent-1 Segregation

- In anaphase I, one normal and one translocated chromosome are moved to one cell pole while the other normal and translocated chromosome go to the opposite poe. - None of the gametes formed by this pattern is viable because of duplications and deletions of genetic information.

DNA Structure

- Individual nucleotides are assembled into a polynucleotide chain by the enzyme DNA polymerase, which catalyzes the formation of a phosphodiester bond between the 3' hydroxyl group of one nucleotide and the 5' phosphate group of an adjacent molecule. - Two of the three phosphates of a dNTP are removed (as a pyrophosphate group) during phosphodiester bond formation, leaving the nucleotides of a polynucleotide chain in their monophosphate form. - Each polynucleotide chain has a sugar-phosphate backbone consisting of alternating sugar and phosphate groups throughout its length. - Double-helical structure in which the nucleotides repeat every 0.34nm and the helix makes one turn every 3.4nm or 10 nucleotides. - The two strands are antiparallel in orientation. Antiparallel orientation of complementary strands brings the partial charges of complementary nucleotides into alignment to form hydrogen bonds. If complementary strands were to align in parallel, the charges of complementary nucleotides would repel and no hydrogen bonds would form. - Purines have two rings, while pyrimidines have one.

Frameshift Mutation

- Insertion or deletion of one or more base pairs in the coding sequence of a gene leads to addition or deletion of mRNA nucleotides. This can alter the reading frame of the codon sequence, beginning at the point of mutation. - The result would be a frameshift mutation, in which the mutant polypeptide contains an altered amino acid sequence from the point of mutation to the end of the polypeptide. - In addition to producing the wrong amino acids in a portion of the polypeptide, frameshift mutations also commonly generate premature stop codons that result in a truncated polypeptide. - For these reasons, frameshift mutations usually result in the complete loss of protein function and thus produce null alleles.

Deletion - Unpaired Loop

- Irrespective of the mechanism that produced them, prophase I homologous chromosome synapsis during meiosis produces a telltale signature of partial chromosome duplication or deletion. - Homologous pairs that are mismatched because one contains a large duplication or deletion will form an unpaired loop in synapsis. - Along most of the length of the homologous pair, normal synaptic pairing occurs. But in regions of structural difference, the extra material present on one chromosome bulges out to allow synaptic pairing on either side. - The material in the loop is normal genetic material of one chromosome carries a deletion, and it is duplicated genetic material if one homolog carries a duplication.

Telomeres

- Linear chromosomes cannot be replicated all the way to their ends. Instead, eukaryotic chromosomes get progressively shorter with each replication cycle. - This is a consequence of an RNA primer being located at one end of the lagging strand and thus not able to be replaced by DNA. In consequence, the resulting lagging strand is shorter than its template strand, causing the chromosome to become shorter with each replication cycle. - The problem is solved by the presence at chromosome ends of repetitive DNA sequences called telomeres. They do not contain protein-coding genes but instead are made up of repeats that are most often 6-bp sequences repeated hundreds or thousands of times. Since its sequences are repetitive and contain no genetic information, portions of the telomere can safely be lost in each replication cycle. - Genetic mechanisms monitor telomere length - one the shortening reaches a critical point, the cell is directed into the apoptotic pathway.

Intergenic Suppressor Genes - tRNAs

- Many intergenic suppressor genes function in mRNA translation. - Each suppressor gene works on only one type of nonsense, missense or frameshift mutation. - Suppressor genes often encode tRNAs, which possess anti-codons that recognize stop codons and insert an amino acid. - Three classes of tRNA nonsense suppressors exist, one for each stop codon (UAG, UAA, UGA). - tRNA suppressor genes coexist with wild type tRNAs. - tRNA suppressors compete with release factors, which are important for proper amino acid chain termination. - Small number of read-through polypeptides are produced; tandem stop codons (UAGUAG) are required to result in correct translation termination.

Site-Specific In Vitro Mutagenesis

- Method by which mutant alleles can be synthesized in the lab and transformed into cell culture and animals. - Commonly used to study mutations of human genes in mice or other model organisms. - An oligonucleotide with the desired sequence change is used to alter a single-stranded DNA sequence. This altered DNA sequence is then converted into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro DNA synthesis. - Use of the uracil-containing template allows rapid and efficient recovery of mutants.

A-Form DNA

- More compact than B-form with about 11 base pairs per complete helical twist and a higher degree of tilt of the base pairs relative to the backbone. - Occasionally detected in cells.

DNA Repair in Eukaryotes

- Most eukaryotic repair mechanisms excise lesions from DNA. Two major pathways are base excision repair (BER), which eliminates single damaged-base residues, and nucleotide excision repair (NER), which excises damage within oligomers that are 25-32 nucleotides long. - Eukaryotes also have mismatch repair, but it is not clear how old and new DNA strands are identified. - Four genes are involved in humans: hMSH2 (homologous to E. coli mutS), hMLH1, hPMS1, and hPMS2 (all homologous to mutL). - All of these are mutator genes, and mutation in any one of them confers hereditary predisposition to hereditary nonpolyposis colon cancer. - Some human genetic diseases result from defects in DNA replication or repair: Xeroderma pigmentosum is an example. The defect is in excision repair, and the inability to repair radiation damage to DNA often results in malignancies. Affected individuals are photosensitive, and portions of skin exposed to light show intense pigmentation and warty growths that may become malignant.

Position Effect Variegation

- Moving a gene near centromeric heterochromatin silences its activity in some cells and not others. - Can occur as a result of inversion.

Suppressor Mutations

- Mutations that occur at sites different from the original mutation and mask or compensate for the initial mutation without reversing it.

Deaminating Agents

- Nitrous acid (HNO2) is a deaminating agent, meaning an agent that removes an amino group (NH2). - It is capable of removing amino groups from any nucleotide. - In most instances, deamination produces no mutagenic effect; but deamination of 5-methylcytosine is an exception, leading to G-C to A-T base-pair substitution. - In addition, when nitrous acid deaminates adenine, the product is hypoxanthine, a modified nucleotide that can mispair with cytosine and lead to an A-T to G-C base-pair substitution mutation.

Spontaneous Mutations

- Occur without a known cause (accident, metabolic by-products, unknown environmental mutagen), and the rate of occurrence differs by species and by gene. - They arise primarily through errors during DNA replication and through spontaneous changes in the chemical structure of nucleotide bases. - There is usually about one new mutation per human birth.

Chromosome Inversion

- Occurs as a result of chromosome breaks followed by reattachment of the free segment in the reverse orientation. - Inversion most commonly affects just one member of a homologous pair and such organisms are either paracentric or pericentric inversion heterozygotes in which one chromosome has normal structure and the homolog contains an inversion. Inversion heterozygotes may experience no genetic or phenotypic abnormalities, as long as no critical genes or regulatory DNA sequences are disrupted by chromosome breaks.

Oxidative Reactions

- Oxidation is a chemical process of electron transfer by addition of an oxygen atom or removal of an atom of hydrogen. - Numerous oxidizing agents such as bleach and hydrogen peroxide cause oxidative reactions that can lead to mutations. - Ex: The oxidized form of guanine frequently mispairs with adenine, leading to a transversion mutation (G-C to T-A).

Radial Loop-Scaffold Model

- Predicts that the chromatin loops gather into rosette-like structures and are further compressed by nonhistone proteins. - The total compaction of chromatin achieved by metaphase is approximately a 250-fold compaction of the already condensed 300-nm fiber.

Griffith Experiment

- Provided indirect evidence that DNA is the molecule responsible for conveying hereditary characteristics in bacteria. - Griffith studied strains of the bacterium Pneumococcus. He found that strains of the bacterium that cause pneumonia in mice grow in colonies that have a smooth (S) appearance, whereas those Pneumococcus strains that do not cause disease are identifiable by their rough (R) appearance. It was later determined that rough bacterial strains have a mutant allele of the polysaccharide gene, which results in a weakened and easily broken capsule. This single gene mutation thus leaves R bacteria vulnerable to attack by mouse immune system antibodies. - The S and R forms of Pneumococcus occur in four antigenic types of the bacteria, identified as I, II, III and IV. Each antigenic type elicits a different immune response from the mouse immune system as a result of the presence of several genetic differences. A single mutation of the polysaccharide gene can convert an S strain to an R strain of the same antigenic type - for example, converting an SII strain to an RII strain. - His most important observations are derived from four injection tests he performed using S and R bacterial strains of different antigenic types. Results: 1. Injecting mice with S-strain bacteria produces illness and death. 2. Injection of "heat-killed" S-strain bacteria does not induce illness. 3. Injection of an R strain does not produce illness. 4. Injection of a mixture of heat-killed SIII strain and living RII strain resulted in mouse death by SIII infection. - Knowing that this outcome could not have been the result of a simple mutational event, Griffith proposed that a molecular component he called the transformation factor was responsible for transforming RII into SIII. - Now we know that the process identified by Griffith is a naturally occurring process called transformation, which is used by bacteria to transfer DNA.

Nucleotide Excision Repair

- Recognized and removes bulky adducts that distort DNA. - Targets bulky modifications generated by the action of alkylating agents and distortions caused by UV light. - Repair of the damage involves enzymes that recognize and bind to the damaged region, followed by removal of a short segment of as many as several dozen nucleotides from the damaged strand. - The missing nucleotides are replaced by the activity of DNA polymerase followed by that of DNA ligase.

Angelman Syndrome

- Results in delayed development, intellectual disability, severe speech impairment and problems with movement and balance (ataxia). - Affects both sexes equally. - AS critical region is between 11q and 13q region of maternal chromosome 15. - Mainly caused by maternal deletion in the 11q-13q region of chromosome 15 - 70%. - Maternal mutation in a specific gene (UBE3A) - 11%. - Paternal disomy - both chromosome 15s are inherited from the father and no chromosome 15 is present from the mother - 2%. - Maternal imprinting defect - 3%.

R-Banding

- Reverse of G-banding. - Obtained by incubating the slides in hot phosphate buffer, then a subsequent treatment of giemsa dye. - The dark regions of the chromosomes tend to be euchromatic and rich in guanine and cytosine whereas the light regions tend to be heterochromatic and rich in adenine and thymine.

Hershey & Chase Experiment

- Showed that DNA, and not protein, is responsible for bacteriophage infection of bacterial cells. - Bacteriophages are viruses that infect bacteria. Phages such as T2, for example, consist of a protein shell with a tail segment that attaches to a host bacterial cell and a head segment that contains DNA. T2 phages are among the many bacteriophages that do not carry any RNA. Infection by a phage culminates in the lysis of the host cell and the release of dozens of progeny phases. - Proteins contain large amounts of sulfur, but almost no phosphorous; conversely, DNA contains a large amount of phosphorous but no sulfur. - Hershey and Chase initially grew phage cultures in different growth media. One growth medium contained S35, the radioactive form of sulfur to label protein, and the other contained radioactive phosphorous P32 to label DNA. The researchers used radioactively labeled phages from each medium to infect unlabeled host bacterial cells in parallel experiments. - After a short time, each mixture was agitated in a blender to separate bacterial cells from the now empty phage shells (ghosts). The relatively large bacterial cells were easily separated from the ghosts by centrifugation. The heavier bacteria collect in a pellet at the bottom of the centrifuge tube, while the lighter ghosts remain suspended in the supernatant. - Testing each fraction for radioactivity revealed that virtually all the P32 label was associated with newly infected bacterial cells and almost none with ghost particles. On the other hand, the S35 label was found in the ghost-particle fraction, and only trace amounts were found associated with the bacterial pellet. - This result demonstrates that phage DNA, but not phage protein is transferred to host bacterial cells and directs the synthesis of phage DNA and proteins, the assembly of progeny phage particles and ultimately the lysis of infected cells.

Tautomeric Shifts - Results

- Tautomeric shifts can lead to base-pair substitution mutations. - For example, consider: the rare tautomer of thymine mispairs with guanine during DNA replication. A tautomeric shift of thymine switches it back to its common, more stable form, leaving a base-pair mismatch of T-G. - If this mismatch is not repaired, DNA replication produces one chromatid with a wild-type T-A base pair and the sister chromatid with a C-G base pair substitution mutation. - This is a transition mutation: G replaces A and T replaces C.

Telomerase

- Telomeres are synthesized by the ribonucleoprotein telomerase, consisting of several proteins and a molecule of RNA. - The telomerase RNA molecule is encoded by a distinct gene and acts as the template for the telomeric DNA repeat sequence. - Telomerase is active in germ-line cells (to ensure that gametes pass on full-length chromosomes) and in some stem cells (to ensure that cells that differentiate from those cells have full-length chromosomes), but it is virtually inactive in differentiated somatic cells. - Hayflick limit - the number of cell divisions of cultured cells is dependent on the source of the cells. - Mutation that limits telomerase activity induces premature aging, while mutations that increase telomerase activity lead to cancer.

30-nm Fiber

- The 10-nm fiber is an unnatural state for chromatin. - Under normal cellular conditions, chromatin forms the 30-nm fiber, which is six times more condensed than the 10-nm fiber. It is produced by coalescence of the 10-nm fiber into a cylindrical filament of coiled nucleosomes that is hollow in the middle - a solenoid structure. - Each turn of the solenoid structure contains 6-8 nucleosomes with a diameter of approximately 34nm. - Chromatin exists in a 30-nm-fiber state or a more condensed state during interphase.

Imprinting - Prader-Willi Syndrome

- The insulin growth factor 2 (IGF2) and H19 genes are encoded very near one another on chromosome 15. - IGF2 on the paternally derived copy of the chromosome is expressed, whereas the IGF2 gene on the maternally derived chromosome is silent. The opposite is the case for the H19 gene. - The q11-q13 region on paternal chromosome 15 is the PWS critical region. - Prader-Willi Syndrome most often results from partial deletion of the portion of the paternal copy of chromosome 15 containing H19 and IGF2 (segment 11-13 on the q arm) - 70% - The condition can also occur if the paternal chromosome 15 is not properly imprinted - 5%. - Finally both chromosome 15s may be inherited from the mother (maternal disomy) and no chromosome 15 is present from the father - 25%. - Characterized by severe hypotonia and feeding difficulties in early infancy, followed in later infancy by excessive eating.

Interstitial Deletion

- The loss of an internal segment of a chromosome that results from two chromosome breaks. - Ex: WAGR syndrome and WAGRO syndrome. Both result from an interstitial deletion in the p arm of chromosome 11. The initials stand for Wilms tumor (hereditary kidney cancer), aniridia (absence of the iris in the eye), genitourinary abnormalities, mental retardation and obesity. Patients with the largest deletions have all five conditions, whereas patients with smaller deletions may have just one or two of the disorders.

Depurination

- The loss of one of one of the purines, adenine or guanine, from a nucleotide by breakage of the covalent bond at the 1' carbon of deoxyribose that links the sugar to the nucleotide base. - Living cells lose thousands of purines a day, making depurination one of the most frequent spontaneous chemical changes affecting DNA. - This forms a DNA lesion known as an apurinic (AP) site. Nearly all AP sites are replaced by the correct purine before the next DNA replication cycles. Repair enzymes use the opposite strand to identify the correct complementary purine to fill the AP site. - If an AP site is not repaired before the beginning of the next replication cycle, the site does not contain a nucleotide that can act as a template base. DNA polymerase will then place a nucleotide, most commonly adenine, opposite the AP site. The strand with the inserted adenine goes on in the next replication cycle to direct the addition of thymine to its daughter strand and the mutation has been generated. This is a transition mutation with T replacing G and A replacing C. - The AP site might be repaired before the next replication cycle, but if not, then during the next DNA replication cycle, it once again is likely to attract an adenine to fill the newly synthesized strand.

UV Radiation & Mutagenesis

- The mutagenicity of UV radiation derives from specific lesions it creates in DNA. - UV irradiation alters DNA nucleotides by inciting the formation of additional bonds that form aberrant structures called photoproducts. - One type of photoproduct - a pyrimidine dimer - is produced by the formation of one or two additional covalent bonds between adjacent pyrimidine dinucleotides in a strand of DNA. - One is a thymine dimer, which has two covalent bonds joining the 5 and 6 carbons of thymines that are adjacent in the same DNA strand. - Its formation distorts DNA by pulling the dimerized nucleotide bases closer together and disrupting hydrogen bond formation with their complementary nucleotides on the opposite strand. DNA replication can thus be disrupted.

Base-Pair Substitution

- The replacement of one nucleotide base pair by another.

DNA Replication - Termination

- To complete DNA replication, RNA primers must be removed and replaced with DNA and Okazaki fragments must be joined together to form complete DNA strands. - In E. coli these tasks are accomplished by the enzymes DNA polymerase I and DNA ligase that are each part of the replisome complex at each replication fork. - When DNA pol III on the lagging strand reaches an RNA primer, thus running out of template, it leaves a single-stranded gap between the last DNA nucleotide of the newly synthesized daughter strand and the first nucleotide of the RNA primer. The pol III, having very low affinity for these DNA-RNA single-stranded gaps, is then replaced by pol I, which has high affinity for such gaps. The DNA pol I removed nucleotides of the RNA primer one by one and replaces them with DNA nucleotides, beginning with the 5' nucleotide of the RNA primer and progressing in the 3' direction until all the the RNA nucleotides in the primer have been replaced by DNA nucleotides complementary to the template strand. - Once the entire RNA primer is replaced, a remaining single-stranded gap sits between two DNA nucleotides. At this point, DNA ligase, having exclusive and very high affinity for DNA-DNA single-stranded gaps, is attracted to the gap and there performs its single task of forming a phosphodiester bond between the two DNA nucleotides that joins two Okazaki fragments.

Chromatin

- To manage the massive amount of DNA and the multiple chromosomes and need for periodic chromosome condensation, eukaryotic chromosomes are organized by a nucleoprotein complex known as chromatin - a mixture of the DNA that makes up the chromosomes along with an array of proteins that organize and compact the DNA.

Meselson-Stahl Experiment

- Took advantage of the newly developed method of high-speed cesium chloride (CsCl) density gradient ultracentrifugation to decipher the mechanism of DNA replication. - A tube filled with a CsCl mixture is subjected to high ultracentrifuge speeds that exerts thousands of gravities of separating force, creating a density gradient throughout the CsCl mixture. When substances are placed in the CsCl gradient and ultracentrifugation takes place, the substances migrate until they reach the point in the density gradient where their molecular density is matched by that of the gradient. - Meselson and Stahl began their experiment by growing E. coli in a growth medium containing the rare heavy isotope of nitrogen - N15 - for many generations. Under these growth conditions, parental DNA is fully saturated with heavy-isotope-containing nitrogen. All of the DNA duplexes contain only the heavy nitrogen isotope, and they are designated N15/N15 to signify the incorporation of N15 in both strands of the duplex. - DNA collected for CsCl gradient analysis from this starting generation, designated generation 0, was exclusively N15/N15. - Next, some of these labeled E. coli were transferred to a new growth medium containing only the normal light isotope of nitrogen, N14. - At the end of each successive DNA replication cycle, DNA was collected from a few cells on the N14 medium for CsCl analysis. Growth in this medium leads to the incorporation of DNA nucleotides containing the light isotope into newly synthesized strands.

Ames Test

- Used to test the mutagenic potential of natural and synthetic compounds. - Exposes bacteria to experimental compounds in the presence of a mixture of purified enzymes produced by the mammalian liver. In animals, ingested chemicals are routed to the liver, where they are broken down by detoxifying enzymes. Using a critical subset of detoxifying liver enzymes called the S9 extract, the Ames test mimics the biological defense processes that take place in the liver of animals exposed to chemical compounds. During enzymatic breakdown in the liver, numerous intermediate products can be produced, some of which may be mutagenic, even if the original compound was not. The purpose of the Ames test is to detect whether the original compound or any of its normal breakdown products are mutagens. - The Ames test most commonly uses strains of the bacterium Salmonella typhimurium that carry mutations affecting their ability to synthesize the amino acid histidine. These bacteria are designated his(-) to indicate their mutation prevents histidine synthesis. They will not grow unless they are provided with a medium that is supplemented with histidine. - The Ames test is designed to identify the rate of reversion mutations (his(-) to his(+)) that restore the ability of bacteria to synthesize their own histidine, thus eliminating the need for histidine supplementation of the growth medium. - The Ames test uses multiple his(-) strains of S. typhimurium, each carrying different kinds of mutations of histidine-synthesizing genes. Some test strains carry base-pair substitution (transition and transversion) mutations; other carry frameshift mutations. The use of these different mutant strains allows detection of compounds that induce base-pair substitution mutations as well as those that induce frameshift mutations, by comparing reversion rates in experimental bacterial cultures exposed to potential mutagens with spontaneous reversion rates in control bacterial cultures. - In each experimental culture, the S9 extract is added to mutant strains of S. typhimurium with base-pair substitution or frameshift mutations and each mixture is separately plated onto a medium lacking histidine. The test compound, in different concentration, is added to a filter paper disk in the center of each test plate, and the plates are incubated. To determine the frequency of containing his(-) bacteria and S9 are plated onto medium lacking histidine and the plates are incubated. Bacteria on the control plates are not exposed to the test compound. - The results of an Ames test are interpreted by counting the number of growing colonies on each plate and comparing the numbers to one another and to the control plates. These data are used to determine whether a test compound is mutagenic and to develop a growth-response curve describing how the concentration of the test compound affects its mutagenicity. - A positive result, indicating that the test compound is mutagenic, is indicated by a significant increase in the reversion rate in bacterial cells that have one kind of mutation over cells that have the other kind of mutation and over the spontaneous reversion rate.

Chromosome Banding

- Using chromosome staining methods and microscopy, scientists can distinguish each chromosome by its overall size and shape and by the patterns of light and dark chromosome banding that are produced along the length of chromosomes by treatment with specific dyes and stains. - Involves arresting the cycle in or just before metaphase. - Used to locate genes, to analyze differences between species and to diagnose genetic diseases.