AGT Lab Manual Information
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| Nucleus | Membrane, Chromatin, and Nucleoli
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| Nuclear Matrix | Fibrous material that remains if the chromatin and nucleoli are extracted
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| Heterochromatin | Highly condensed chromatin that stains darkly with nuclear stains
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| Euchromatin | More dispersed chromatin which stains lightly or not at all
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| Diameter of DNA fibers with proteins | 30nm
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| Diameter of protein depleted DNA fibers | 10nm
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| Primary functions of the nucleic acids | gene replication (the process of copying sequences of DNA(genes) for distribution to daughter cells) and gene transcription ( the process of copying sequences of DNA into complementary strands of RNA)
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| Occurrence of protein synthesis | cytoplasmic ribosomes
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| Nuclear envelope | porous double membrane surrounding the nucleus with ribosomes attached to the outside; disappears during cell division; contains the nucleolus and the chromatin
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| Nucleolus | 1-4 in the nucleus; site of ribosome precursor assembly; formed by NOR of acrocentric chromosomes; composed of RNA protein, and some DNA
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| Chromatin | composed of DNA, proteins (mainly histones), RNA, and certain polysaccharides
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| DNA | double helix of two strands composed of nucleotides
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| Nucleotide formation | sugar molecule, phosphate groups, and one of four bases: adenine, guanine, thymine, or cytosine
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| Hydrogen Bonds | hold the bases of the two strands together
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| 5-methyl cytosine | often concentrated in areas of heterochromatin, such as in 1, 9 , 15, 16, Y
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| Semiconservative replication | two identical copies of the original DNA composed of one original strand and one newly synthesized strand
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| Codon | triplet code of three bases that codes for one amino acid
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| Gene | a linear arrangement of codons giving the instructions for the building of a protein with specific amino acids in a particular order
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| Missense mutation | one that changes a codon specific for one amino acid to one that specifies a different amino acid
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| Nonsense mutation | single base substitution in DNA resulting in a chain termination codon
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| Introns | DNA sequences not present in the mRNA and are not translated into amino acids in that gene's protein
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| Exons | sequences present in the mRNA that usually code for protein
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| Denaturation to two single-stranded helices | High temperature or high-PH will break the hydrogen bonds, unwinding or denaturing the DNA
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| Hydrogen bonds | G-C pairs have three; A-T have two (tend to be more unstable and will denature before G-C)
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| Tm | temperature at which a given DNA will be half denatured or melted; used as an index of the amount of G and C in that DNA
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| Cot curve | curve of the rate at which denature DNA renatures(Co = concentration of single-stranded DNA; t = time); tells how many sequences are present in multiple copies (repetitive DNA) versus how many are unique
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| Buoyant density of DNA | measure of the G-C content by forming gradients of concentration in cesium chloride during centrifugation; depends on DNA strandedness and base composition
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| RFLPs | Restriction fragment length polymorphisms (ex. beta-globin gene on chromosome 11 and HpaI; 7600bps in some; 13,000bps in others [sickle cell disease])
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| Sickle Cell Disease | HpaI restriction enzyme; MstII restriction enzyme: CCTGAGG, 1150 and 200bp; CCTGTGG for affected producing 1350bp
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| Oligonucleotides | (oligo = few) short molecules engineered to match portions of a normal gene exactly; used to detect genetic defects that involve a point mutation or change in a single base
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| RNA differ from DNA | RNA (DNA): 2'-OH group (2'-H group); single-stranded (double-stranded); uracil (thymine) for adenine
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| Transcription | DNA template for complementary strand of RNA
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| mRNA | complementary strand of template DNA; introns are spliced out; molecule moves out of the nucleus to the cytoplasm
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| tRNA | binds the appropriate amino acid to its anticodon (base triplet complementary to a codon in mRNA)
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| rRNA | ribosomal RNA assists in the actual protein synthesis (anticodons of the tRNA molecule bind the codons of the mRNA molecule)
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| Chromsome proteins | histones and nonhistones
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| Histone protein characterization | contained in interphase chromatin; characterized by their basic pH and large numbers of the amino acids arginine and lysine; isoelectric point always more than 10
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| Nonhistone protein characterization | isoelectric point always less than 10; tend to be acidic (4-9); different structural, enzymatic, and regulatory functions
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| Isoelectric Point | pHs at which the average charge of the molecule is zero
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| Histone protein classes | H1 (lysine-rich); H2A&H2B (slightly lysine-rich); H3&H4 (arginine-rich); H5 replaces H1 in nucleated erythrocytes; Histones are extracted from chromatin by dilute acids or by high-molarity salt solutions; ratio to DNA by weight is 1:1
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| Protamines | low molecular weight basic protein that replaces histones in mature sperm
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| Acetic Acid and Methanol | Fixative solution; dissolves out some, if not most, of the histones
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| Nonhistone proteins | include all proteins of chromatin other than histones; more numerous and more variable, but less of the chromatin mass; involved in chromatin metabolism, gene expression, and in higher order structure
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| Nu-body/Nucleosome | octamer of two molecules of each H2A, H2B, H3, & H4 histones (10nm)
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| H1 histone protein | involved in linking and compaction of nucleosomes
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| Nucleosome | basic unit of eukaryotic chromatin, present in dispersed or condensed chromatin, in repetitive areas of unique sequences, and in interphase and metaphase
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| Heteropyknosis | property of chromatin of showing variations in staining intensity owing to differences in the degree of coiling or condensation of the chromatin filaments
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| Positive Heteropyknosis | Chromosomes and segments of chromosomes that are more heavily stained than the rest
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| Negative Heteropyknosis | Chromosomes and segments of chromosomes that are more lightly stained than the rest
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| Positive Heteropyknosis in G-banding | Heterochromatin
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| Negative Heteropyknosis in G-banding | Euchromatin
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| Facultative heterochromatin | condensed, inactive chromatin of X chromosomes in excess of one; may represent one or the other in a given cell; sequences similar to active DNA, does not stain differently, and can become decondensed and active
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| Constitutive heterochromatin | differentially staining areas of chromatin and chromosomes and are constant from cell to cell; rich in repetitive DNA, stains differently from euchromatin; never elongates or decondenses
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| Heterochromatin | Does not code for protein (genetically inactive) and replicate late in the synthesis phase of the cell cycle
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| Barr body | facultative heterochromatin of the second X chromosome - discovery: 1949 Murray L. Barr and Ewart G. Bertram as a "paranucleus"
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| Russell-Lyon Hypothesis, first part | 1. One of the two X chromosomes is inactivated in human females 2. the inactivated X may have either maternal or paternal origin in a given cell of an individual, and the choice is random
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| Russell-Lyon Hypothesis, last part | 3. inactivation occurs in early embryogenesis 4. inactivation is stable, and descendants of a cell with an inactive X inherit that same X in an inactive state
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| Identifying the inactivated X | BrdU for 40-44 hours; add thymidine 6-7 hours before fixation; stain with Hoeschst 33258; observe with fluorescence microscope; or thymidine, then BrdU, and observe pale-staining X
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| Dosage compensation | X inactivation; mechanism for producing equal amounts of gene products in females having two X chromosomes and males having only one
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| Xq13 | inactivation center of the X chromosome
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| Abnormal X vs Normal X inactivation | The abnormal X is usually inactive as they are more likely to survive than when active in cells
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| X;autosome translocation vs Normal X inactivation | The normal X is usually inactive
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| unbalanced X;autosome trans vs Normal X inactivation | the translocation chromosome is usually inactive and may or may not extend into the autosome
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| 5-azacytidine | produces hypomethylation of DNA
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| XIST | active only on the inactive X chromosome in band q13
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| Satellite DNA | Repetitive DNA found in constitutive heterochromatin; can mean any highly repeated sequences
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| Alpha, beta, and classical DNA | found at the centromeres of all the chromosomes
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| SINES | Short interspersed elements located in the quinacrine pale bands; containing cleavage sites recognized by the restriction endonuclease AluI
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| LINES | Long interspersed elements located in the quinacrine bright bands; cleavage sites for L1
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| Microsatellites | di- or trinucleotide tandem repeats and are highly polymorphic
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| SSRs (simple sequence repeats) | 3- to 6-bp repeats found in coding and noncoding DNAs and are highly polymorphic
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| Minisatellites | Longer repeats, usually more than 10bp, and usually located at the distal ends of chromosomes, and are highly polymorphic, useful for DNA fingerprinting
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| Classification of Chromosomal Sequences | Beta --> Alpha --> Classical --> Telomeric --> Unique Gene --> Total chromosome
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| Cell cycle schedule | 18 hrs: 9 hr for G1; 5 hr for S; 3 hr for G2; and 1 hr for M
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| S cycle inhibitors | amethopterin (methotrexate); hydroxyurea; and cytosine arabinoside
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| Mitotic arrestants | Vinca alkaloids; colchicine (alkaloid); and podophyllin
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| Prophase | Nucleolus disperses throughout the nucleus and the centrioles migrate to opposite poles
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| Prometaphase | Nuclear envelope breaks down in most organisms
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| Five stages of Prophase I of Meiosis I | Leptonema, Zygonema, Pachynema, Diplonema, and Diakinesis
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| Leptotene Stage | Nuclear chromatin begins to condense, but chromosomes are not yet evident, the telomeres are attached to the nuclear envelope, and the cell has an enlarged nucleus and finely dispersed chromatin
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| Zygotene Stage | Synapsis (homologous chromosomes lie next to and attach to each other) occurs (bivalents), synaptonemal complex forms where crossing over can occur, and the sex vesicle can be seen
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| Pachytene Stage | Bivalents shorten and become thick, crossing over occurs, and chromomeres of the G-band patterns of mitosis prometaphase chromosomes can be discerned; bivalents are known as tetrads
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| Diplotene Stage | Nucleolus detaches from its associated bivalents and bivalent chromosomes begin to separate as their centromeres pull them apart, but are still attached at chiasmata (sites of crossing over); end of sex vesicle
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| Diakinesis Stage | Occurs in males: chiasmata move toward ends of the bivalents, nucleolus dissipates, and nuclear envelope disappears; females: meiosis halted in diplotene or dictyotene stage until ovulation
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