Bio3400 Chapter 12 DNA Organization and Chromosomes
  1. Bacterial and        chromosomes usually consist of a single molecule, are much smaller than eukaryotic chromosomes, and largely devoid of associated           .

      Viral chromosomes can be either DNA or RNA, and either single or double stranded. They can be circular or linear molecules, and are tightly packed in a protein coat. Bacterial chromosomes are always circular, double-stranded DNA molecules, compacted into a structure called the nucleoid.


      The bacteriophage T2 has a double stranded, linear DNA genome. The nucleotide sequences of individual viruses are circular permutations of a common sequence.


      The bacteriophage l (lambda) possesses a linear double-stranded DNA molecule in its protein coat, which closes to form a ring upon infection of the host cell, as seen in the electron micrograph at right.


      Bacteriophage T4 is one of a group of lytic (or virulent) bacterial viruses called T-even phages. The "head" is made up of an protein coat containing the DNA. A "tail" contains a collar and a contractile sheath surrounding a central core; tail fibers protruding from a base plate contain binding sites that recognize the cell wall of the E. coli host.


      Bacteria such as Escherichia coli have a circular, double-stranded DNA chromosome. The chromosome can be visualized under the electron microscope if the cell is lysed in a hypotonic medium.
     
     
     
     
  2. Bacterial and viral chromosomes are often           and underwound, resulting in a compact,              conformation.

      A normal double helix can form an energetically relaxed circle with 20 right-handed turns (linking number L = 20). continue


      If such a circular molecule were underwound by 2 turns (L = 18), the resulting circle is energetically strained, and will spontaneously form two negative (left-handed) supercoils, restoring the number of original turns. The supercoil is more compact, and most circular DNA molecules in bacteria and phages are supercoiled.
     
     
     
     
  3. Distinctive banding patterns seen in           chromosomes represent paired homologs; bands may exhibit        indicating gene activity.

      Giant polytene chromosomes, found in some fly larvae, represent paired homologs. Each chromosome undergoes many rounds of DNA replication, but without strand separation or cell division. Over 1000 strands may remain attached to one large centromere, exhibiting banding patterns under a light microscope.


      Polytene chromosomes. DNA strands in bands (B) can uncoil, or puff (P), during transcription. Thus bands indicate the presence of genes, while interband (IB) regions are devoid of genes.
     
     
     
     
  4.            chromosomes in eukaryotes possess extensive DNA        where transcription activity can be visualized.

      Lampbrush chromosomes are found in most vertebrate oocytes, when they uncoil during transcription. The decondensed loops are composed of one DNA double helix, active in transcribing RNA.
     
     
     
     
  5. Eukaryotic DNA is complexed with          proteins and bound up in repeating units called              .

      In eukaryotic chromatin, DNA is associated various proteins, including histones and nonhistones. Histones are proteins rich in positively charged amino acids (lysine and arginine) bound to the negatively charged phosphate groups of nucleotides and function to pack 2-m long lengths of DNA into a 10-Ám diameter nucleus.


    • The distance between nucleotides is 0.34 nm, or .34 x 10^-9 m = 3.4 x 10^-10 m.
    • You have about 6 billion base pairs per cell in 46 chromosomes (p. 264).
    • (6 x 10^9) x (3.4 x 10^-10 m.) = 2.04 m. of DNA per cell.
    • You have about 200 trillion (2 x 10^14) somatic cells.
    • If all the DNA in your cells were uncoiled and lined up end-to-end, their total length is (2 x 10^14) x 2 m. = 400 trillion m. = 400 billion km.
    • The distance from Earth to the Sun is about 150 million km, or 300M km round trip.
    • You have enough DNA to take over 1,000 round trips to the Sun. Updated Sep 12, 2007 by Peter Chen


      Each nucleosome consists of a core of 147 base pairs of DNA coiled around 2 tetramers of histone proteins. Nucleosomes are linked together via short segments of spacer DNA and H1 histone into repeating units of about 200 base pairs.


      The repeating units of nucleosome resemble "beads on a string". Digestion with a nuclease produces 200 base-long particles of nucleosomes where the DNA is protected by its associated histone proteins.
     
     
     
     
  6. The nucleosomes are condensed in several levels of DNA packing to form the compact             packed into the 10-µm nucleus.

    • In the first level of DNA packing, A 2-nm diameter DNA molecule is coiled around 2 tetramers of histone proteins into a nucleosome that is about 11 nm in diameter. Histone H1 acts as a spacer between nucleosomes. continue


    • The 11-nm fiber is packed into a 30-nm solenoid consisting of several nucleosomes coiled together.
    • During condensation to the mitotic chromosome, the 30-nm fiber forms a series of looped domains that further condense into a 300-nm chromatin fiber.
    • The 300-nm fibers then coil into the chromatid arms seen in metaphase chromosomes.
     
     
     
     
  7. Several           techniques can be used to reveal          patterns in mitotic chromosomes that are useful in identifying chromosomes.

      Chromosome preparations can be heat denatured and treated with Giemsa stain. Condensed and inactive DNA regions, called heterochromatin, pick up the stain, as shown in these heterochromatin regions near the centromere .


      If mitotic chromosomes are treated with the proteolytic enzyme trypsin followed by Giemsa staining, a G-banding pattern emerges that can be used to identify chromosomes and chromosomal mutations.


      The G-banding patterns can be used to identify chromosomes and regions on each chromosome. This X chromosome shows the banding nomenclature along its p ("petite") and q ("queue") arms.


      Errors in crossing over or chromosome breaks may cause chromosome rearrangements. A chromosome break may produce "sticky ends" that can rejoin other broken ends. The rejoined chromosome may exhibit deletions, duplications, inversions, and translocations.
     
     
     
     
  8. Eukaryotic chromosomes often exhibit large amounts of             DNA.


        Repetitive DNA can be grouped into many categories.
       
       
       
       
    • Repetitive DNA includes            DNA and              DNA that mediate chromosomal migration.

        Most eukaryotic DNA exhibit uniform density (indicating similar G-C/A-T rations), and are detected in a main band when analyzed with sedimentation equilibrium centrifugation. Often, a satellite band with a different density is also observed, containing repetitive DNA sequences.


        Insedimentation equilibrium centrifugation (or density gradient centrifugation), different molecules in the mixture will settle in bands of different buoyant densities where the centrifugal force is equal and opposite to the upward diffusion force. The gradient is eluted from the tube in fractions, which can then be measured for UV absorption at 260 nm. This technique can be used to analyze base composition of double-stranded DNA.


        centromeric DNA. Satellite DNA is highly repetitive, and exhibits fast reassociation kinetics. They are found in heterochromatic regions near the centromeres, and can be localized on an autoradiograph by in situ hybridization with a radioactive probe.


        Centromeric DNA (CEN) of yeast is about 125 bp and can be divided into 3 regions. Region II is made of highly repetitive DNA that is abnormally rich in A-T base-pair composition.
       
       
       
       
    • The ends of linear eukaryote chromosomes contain            DNA sequences that play a role in the normal process of cell aging.


      Telomeric DNA at the ends of linear eukaryote chromosomes consists of short tandem repeats: the sequence TTGGGG is repeated many times. They are synthesized by the enzyme telomerase.


      The enzyme telomerase can synthesize short DNA sequences (telomeres) at the 3' end of eukaryotic chromosomes, preventing chromosome shortening in germ cells. The enzyme adds repeats of TTGGGG sequences that fold back on themselves by forming unorthodox G-G hydrogen bonds. The gap is filled by a DNA polymerase and ligase. The hairpin loop is then cleaved off, preserving the original duplex. This allows gametes and malignant cells, as well as some "immortal" cultured cells, to continue duplicating the linear DNA.