text pages 144-151 and 326-331
Internal Organization of the Nucleus
Nucleus has internal structure that organizes the genetic material and localizes some nuclear functions to discrete sites.
Nucleolus - site at which the rRNA genes are transcribed and ribosomal subunits are assembled.
Additional organization suggested by the organization of chromosomes and by the potential localization of functions such as DNA replication and pre-mRNA processing to distinct nuclear domains.
The DNA of eukaryotes is quite complex.
It is organized into morphologically distinct units called chromosomes.
Each chromosome contains a single enormous DNA molecule.
DNA is made compact by a hierarchy of different types of folding, each of which is mediated by one or more protein molecules.
First level
DNA is bound to basic proteins known as histones [contain basic amino acids].
DNA with bound histone is termed chromatin.
Five classes of histones:
H1
H2A
H2B
H3
H4
Histones are extremely rich in the positively charged basic amino acids lysine and arginine. The positive charge of the histones is one of the major features of the molecules, enabling them to bind to the negatively charged phosphates of the DNA.
The histones are highly evolutionarily conserved. Especially H3 and H4
Less so with H1.
H4 from cows differs by only 2 amino acids from H4 of peas. What you can conclude from this is that the structure/sequence of the histones (and therefore function) has not diverged in the ~ 10^9 years since plants and animals diverged.
The appearance of DNA complexed to histones : beads on a string
The beadlike structures are called nucleosomes.
Each nucleosome core (octameric protein disc) is found to consist of:
Two molecules each of histones
H2A
H2B
H3
H4
Around this core of histones a 146 base pair segment of DNA which is wrapped like a ribbon (1.75 times). DNA (linker DNA links nucleosomes one to another) 146 base pairs in association with nucleosome pretty consistent from organism to organism. The linker DNA does vary quite a bit in length.
H1 is then bound to the histone octamer and to linker DNA between nucleosome cores. When H1 binds, there are two full turns of the DNA (166 base pairs) around the nucleosome core particle. In this conformation the structure is referred to as a chromatosome.
The resulting chromatin fiber is approximatley 10nm in diameter. Overall length of DNA has been shortened 6X at this point.
See figure in text.
The H1 binds and pulls the chromatosomes together by coiling into fibers which are approximately 30 nm thick. This structure is termed a 30nm chromatin fiber.
see figure in text
H1 can be removed and the beads on a string structure stays intact but the 30 nm fiber structure is lost. Saccharomyces lacks H1 and its DNA does not form the 30nm chromatin fiber structure.
H1- is composed of an amino terminal arm, a globular central portion, and a carboxyl terminal arm.
To give you an idea of conservation of the process. Histones will even package bacterial DNA into nucleosomes in vitro! Doesn't occur naturally in bacterial cells because there is no histones.
The 30 nm chromatin fibers are further condensed by looping. Even further condensation occurs during the transistion from chromatin to the highly condensed metaphase chromosome. This transition is less well understood.
As cell passes through its growth cycle, chromatin structure changes. In a non-dividing cell the chromatin is dispersed and fills the entire nucleus. The euchromatin is believed to be primarily in the 30nm chromatin fiber form. These fibers are further organized into large loops. ~ 10% of the euchromatin is in more relaxed (decondensed) state [10 nm chromatin fiber] and this is the DNA that is most transcriptionally active.
However, it is not randomly dispersed. Although interphase chromain appears to be uniformly distributed, the chromosomes are actually arranged in an organized fashion and divided into discrete functional domains that play an important role in regulating gene expression. The chromosomes are closely associated with the nuclear membrane at many sites. Individual chromosomes occupy distinct territories within the nuclei .
Cell Cycle
G1 - period between last mitotic division and start of DNA synthesis. Active period of gene transcription.
S- DNA is replicated
G2- cell further prepares to divide
M- Nuclear division (mitosis)
mitosis: prophase, metaphase, anaphase, and telophase
C -Cytoplasmic division (cytokinesis)
During G1 through S, chromatin is dispersed and fills the entire nucleus Makes sense - don't want it to be too condensed for transcription and replication.
Following S and leading up to metaphase of mitosis (100X condensation).
Metaphase chromosomes represent the most condensed phase.
Important because it allows the chromosomes to be faithfully segregated into daughter cells. None of the DNA is transcriptionally active in this conformation.
Centromere - looks like a constriction in the chromosome. Point where two sister chromatids are joined together. Also point of attachment of the spindle fibers during mitosis.
These centromeric regions contain specific DNA sequences and bound proteins. When proteins are bound the structure is termed a kinetochore.
Telomeres- Sequences at the very ends of the chromosome.
Play critical role in chromosome replication and maintenance.
During interphase- some of the chromatin (~10% of total chromatin) remains highly condensed and is termed heterochromatin - this DNA is transcriptionally inactive.
Two types:
constitutive heterochromatin
Contains DNA sequences which are never transcribed [such as highly repetitive satellite DNA sequences].
facultative heterochromatin
Contains sequences that are not transcribed in the particular cell under examination but are transcribed in other cell types. [ex
ample: B lymphocytes are the only cells which express the immunoglobulin genes. During interphase, the Ig genes would be in euchromatin form in B lymphocytes. In all other cell types, the Ig genes would be in the form of heterochromatin.]
The phenomenon of X chromosome inactivation also provides an example of the role of heterochromatin in gene expression. In many animals, including humans, females are XX and males are XY. The X chromosome contains thousands of genes that are absent from Y. Thus, females have twice as many X chromosome genes as males. However, males and female cells have equal amounts of the proteins encoded by the X chromosome. One of the 2 X chromosomes in female cells is inactivated by being converted to heterochromatin early in development.