Genetics Ch. 12
Heterochromatin
Eukaryotic chromatin that remains highly compacted during interphase and is generally not transcribed; replicates later during S compared to euchromain; lacks genes or has repressed genes
CpG islands
Methylation of cytosine within polynucleotide chains of DNA; negatively correlated w/gene activity; creates a 5-methyl cytosine, occuring when next to guanine
Does the space of the head of a virus often exceed the chromosome volume by a factor of two?
No, it seldom exceeds beyond a factor of two
Satellite DNA
a variable proportion of the total DNA that does not have uniform density to the main-band DNA. Highly repetitive DNA (short sequences repeated a large amount of times).
Repetitive DNA
Nucleotide sequences, usually noncoding, that are present in many copies in a eukaryotic genome. The repeated units may be short and arranged tandemly (in series) or long and dispersed in the genome; Three Categories: 1) Heterochromatin associated w/centromeres and telomeres 2) Tandem repeats of both short and long DNA 3) Transposable sequences
Basic Model for Chromatin Structure
1) Chromatin contains repeating units that protects the DNA from enzymatic cleavage except where any 2 units are joined; discovered by use of endonucleases that resulted in fragments of 200bp. 2) EM obs. reveal that they are composed of linear arrays of spherical particles (v-bodies - *nucleosomes*) 3) Repeating units of nucleosomes consist of one of four types of tetramers in association with 200bp 4) Upon extended nuclease digestion, the 200bp are removed from the nucleosome, creating *nucleosome core particles* of 147bp, which becomes responsible for linking nucleosomes, aka *linker DNA*, associated with a fifth histone.
Variable Number Tandem Repeats (VNTRs)
15-199 bp found within and between genes (*minisatellites*); number of these tandem copies at each location varies among individuals, creating localized regions of 1000 to 20k bp, and these regions provided the basis for *DNA fingerprinting*
Middle repetitive DNA
Recognized by reassociation kinetics; includes some duplicated genes, but most are noncoded tandem repeats or interspersed sequences • VNTRs and Microsatellites/STRs
in situ hybridization
A technique using nucleic acid hybridization with a labeled probe to detect the location of DNA or RNA in an intact organism; for satellite DNA studies, used to discover heterochromatic centromeric areas
Puff
bulge result of uncoiling of polytene chromosomes; visible manifestation of high levels of gene activity
bacteriophage lambda
linear double-stranded DNA virus; closes to form a ring upon infection of the host cell
Alphoid family
one of the most recognized satellite DNA sequences in humans found mainly in centromere regions; about 170bp, present in tandem arrays of up to 1 mil. bp; believed to be involved in kinetochore functioning
Position effect
position of a gene or group of genes relative to all other genetic material may affect their expression
Histones
positively charged protein molecules of basic aa's around which DNA is tightly coiled in chromatin
Transposable sequences
sequences interspersed individually throughout genome, not tandem (adjacent); short and long (SINE and LINE) mobile sequences that can move to different locations within the genome; large portion of human genome contains these sequences.
CEN region
the minimal heterochromatic region of the centromere that supports the function of chromosomal segregation; here DNA binds to a platform of proteins called the *kinetochore* that binds to the microtubules making up the spindle fibers during cell division
Linking number (L)
the number of times that two strands of a closed, circular DNA duplex cross over each other, ie. number of turns; 20 turns = L = 20; molecule is energetically relaxed if this number remains the same from linear to circular conformation; if number changes due to a cut/unwinding, molecule is energetically strained; calculate as L = (#bp) / (#bp/turn); ex: L = (5200/10.4) = 500
Epigenetics
the study of modifications of an organism's genetic and phenotypic expression that are not attributable to alteration of the DNA sequence making up a gene
Chromatin
uncoiled DNA
Histone tails
unstructured and not packed into the folded histone domains within the core of the nucleosomes but instead protrude from it; provide potential targets along the chromatin fiber for a variety of chemical modifications that may be linked to genetic functions, inc. chromatin remodeling and regulation of gene expression
Chromatin remodeling
when chromatin is induced to change it's structure to be relaxed to expose regions of DNA to be read
Long Interspersed Elements (LINEs)
• About 6kb long and present 850,000+ times in the human genome • L1 family - 6400bp and present 500k+ times; higly variable 5' end, role in genome undefined, excpet for coding of reverse transcriptase • *Retrotransposons* - similar transposition mechanism to retroviruses • Total 21% of human genome
Chromosome-banding techniques
• Differential staining along longitudinal axis of mitotic chromosome • Staining patterns resemble polytene chromosome bands • C-banding and G-banding
Polytene Chromosomes
• Giant chromosomes consisting of many identical chromatids lying in parallel register or in chromomere linear conformation •Found in salivary, midgut, rectal, and malpighian excretory tubule tissues, larvae of some flies, and many protozoans and plants •Can be seen in nuclei of interphase cells • 200-600µm long • Represent paired homologs, are composed of large numbers of identical DNA strands, undergoing many rounds of replication but without strand separation or cytoplasmic division.
Lampbrush Chromosomes
• Large with extensive DNA looping • Found in most vertebrate oocytes and spermatocytes of some insects • Easily isolated from oocytes in diplotene stage of prophase I of meiosis - active in directing metabolic activities of the developing cell • 500-800µm, revert to 15-20µm in meiosis • Seen as being extended uncoiled versions of normal meiotic chromosomes • Similar to polytene chromosomes puffs
Short Interspersed Elements (SINEs)
• Less than 500 bp long and may be present 500,000+ times in the human genome • Alu family - the presence of DNA sequences identified by restriction endonuclease Alu I; 200-300bp long, dispersed in genome both between and within genes; 5%+ of the human genome; role in genome essential to transposition • Total 13% of human genome
Phosphorylation
• Occurs on aa's of histones via kinases • Post translational; reversible w/right enzyme • Phosphate groups added to -OH groups of aa's S and H • Create a (-) charge on the protein • Correlation to the cycle of chromatin unfolding and condensing during and after DNA replication
Methylation
• Occurs on aa's of histones via methyltransferases • Post-translational; reversible w/right enzyme • Methyl groups added to aa's R and K in histones, correlated w/gene activity
Acetylation
• Occurs to aa's of histones via histone acetyltransferase (HAT) • Post-translational; reversible w/right enzyme • Adding an acetyl group to the positively charged amino group on the side of aa K; provides a (+) charge to the molecule • Linked to gene activation and opening/remodeling of chromatin; example of underaceylation is the other X chromosome (barr body)
Multiple-copy genes
functional genes present in more than one copy
Solenoid structure
30-nm fiber forms when the 10-nm fiber coils into a solenoid structure, with 6-8 nucleosomes per turn and histone H1 stabilizing the solenoid; sixfold increase of compaction of DNA; characteristic of uncoiled chromatin fiber in interphase
C-banding
Chromosomes denature w/heat and treated w/Giemsa stain; only centromeric regions of mitotic chromosomes stained.
Topoisomers
DNA molecules with the same sequence but differ in coiling; two identical molecules but with different linking numbers (L)
Pseudogenes
DNA sequences representing evolutionary vestiges of duplicate copies of genes that have undergone significant mutational alteration; show some homology to parent gene but are usually not transcribed bc of insertions and deletions throughout their structure
G-banding
Differential staining along the length of each chromosomes and involving the digestion of mitotic chromosomes w/trypsin (proteolystic enzyme) followed by Giemsa.
Only a very small amount of the genome codes for proteins. True or False?
True - only about 20k-30k genes code in a sea urchin, less than 10% of their genes; same for Drosophila, only 5-10% is coded • Humans have about 20k functional genes ≈ 2% of our genome
In order to assume a more energetically favorable conformation, an underwound molecule will form supercoils in the direction opposite to that of the underwinding. True or False?
True - two negative (left turn) supercoils are formed re-establishing the number of turns in the helix
Euchromatin
The less condensed form of eukaryotic chromatin that is available for transcription.
HU and H-NS (Histone-like Nucleoid Structuring) proteins
Two DNA-binding proteins associated with bacterial chromosomes that function to fold and bend DNA; small but abundant in cell and contain a high % of (+) charged aa's that can ionically bond to the negative charges of the phosphate groups in DNA
Supercoiled DNA
characteristic of closed-circular molecules; densely packed but base pairing is maintained
øX174 bacteriophage
circular single-stranded DNA virus
Nucleoid
dense region of double-stranded DNA found in prokaryotic cells/bacterial chromosomes
Microsatellites/Short Tandem Repeats (STRs)
di- tri- tretra- penta- nucleotides repeated 5-50 times; dispersed in genome and vary among individuals in # of repeats present @ any site; serve as usefule markers for genome analysis
Topoisomerases
enzymes that change DNA supercoiling by making double-stranded DNA cuts to relieve stress and convert DNA topoisomer conformations; types I and II based on whether they cut one or both of the strands of the helix (respectively); Ex: In E. coli, Topoisomerase I reduces the # of (-) supercoils in a closed circular DNA molecule, but Topo II introduces (-) supercoils into DNA.
