References:

Textbook, pages 407-426

The nucleosome
Packaging of DNA into a metaphase chromosome
Euchromatin, Heterochromatin
Balbiani Rings, Lampbrush Chromosomes, Polyteny
Chromosome Banding
Satellite DNA
Kinetochores
Telomeres
Repetitive DNA

The basic unit of chromosome structure is the NUCLEOSOME. Nucleosomes are separated by short stands of DNA called LINKER DNA. Nucleosomes can be identified by digesting chromosomal DNA with micrococcal nuclease, which will degrade the linker DNA, but not the DNA associated with histones, leaving DNA strands of about 200bp. Further analysis, however, has revealed more of the structural details of nucleosomes.

The basic unit of nucleosome construction is 146 base pairs of DNA wrapped around a core of 8 proteins. The proteins in nucleosomes are HISTONES. There are 4 types of histones in the nucleosomes core, histones H2A, H2B, H3 and H4. A nucleosome consists of 2 of each of these histones, resulting in the 8 proteins of the nucleosome core. The 146 base pairs of DNA in the nucleosome core make 1.70 turns around the histone core. The histone core consists of two dimers and a tetramer. Each dimer consists of an H2A and an H2B histone, while the tetramer consists of 2 H3's and 2 H4's (see figure 15.6a on page 411 of the textbook). Histones are the largest component of chromosomes (see table 15.1 on page 408 of the textbook).

Another histone, histone H1, "locks" the DNA in place around the histone. The core histone associated with histone H1 is called a CHROMATOSOME, and consists of about 168 bp of DNA, 1 histone H1 and 2 each of histones H2A, H2B, H3 and H4. Chromatosomes are separated by 50-75 base pairs of DNA called LINKER DNA (see figure 15.5 on page 411 of the textbook).

Nucleosomes are about 10nm (100 angstroms) in diameter (see figure figure 15.10 on page 413 of the textbook). The first level of packaging of DNA is the nucleosome (chromatosome). The next level of DNA packaging is the coiling of nucleosomes to form 30-nm FIBERS (see figure  15.10 on page 413 of the textbook). The 30nm fiber is sometimes called a SOLENOID.

Between cell division (a phase of the cell cycle called INTERPHASE), two types of chromatin can be observed, EUCHROMATIN (less densely staining) and HETEROCHROMATIN (more densely staining). DNA in euchromatin is mostly in the form of the 10 nm fiber or the 30 nm fiber.  DNA in a 10 nm fiber can be actively transcribed, as RNA polymerase can move through nucleosomes (see figure 15.8 on page 412 of the textbook). About 10% of euchromatin is in the form of the 10 nm fiber.

Heterochromatin is much more condensed than euchromatin, and is not transcriptionally active in the highly condensed form. A third level of DNA coiling found in heterochromatin is the coiling of the 30 nm fiber (see figure 15.11 on page 413 of the textbook) to form a fiber about 240 nm in diameter.

The formation of the highly condensed metaphase chromosomes involves the attachment of the 30 nm fibers (folded or not) to a PROTEIN SCAFFOLD (see figure 15.12 on page 414 of the textbook).

Chromosomes appear to occupy discrete position within the nucleus during interphase with telomeres associated with the cell membrane in at least some cases.

Several structures have been described through the history of genetics, and terms relating to those descriptions have become part of the general language of genetics. The terms we will review today are those associated with the analysis of metaphase chromosomes.

POLYTENY

Polytene chromosomes occur when there is replication of chromosomes but no separation of chromatids. The textbook, on page 414, suggests that polyteny also involves the synapsis of homologs. I do not know if synapsis occurs in polytene chromosomes, but I do know that it is not necessary. Polytene chromosomes can be easily observed under the light microscope as they can consist of thousands of chromatids. Polytene chromosomes are found is cells like the salivary gland cells of insects that produce large amounts of silks and other products of salivary glands. Most of the early studies of chromosomes involved polytene chromosomes in insect, particularly midge flies (Chironomus).

BALBIANI RINGS

"Puffs" can be observed in polytene chromosomes (see figure 15.14 on page 414 and figure 15.15-15.17 on page 415 of the textbook). Such CHROMOSOME PUFFS are often called Balbiani rings. These are regions where the DNA of the thousands of chromatids in a polytene chromosome unwinds to the 10 nm fiber to allow for gene expression. Balbiani rings allow for the first studies of developmental stage specific and/or tissue and even cell specific gene expression through the observation of developmental stage
and/or tissue and even cell specific occurrence of Balbiani rings.

Note figure 15.19 on page 416  of the textbook, where it shows that light bands on chromosomes are due to loosely associated 30 nm fibers and dark bands are due to the more tight coiling of 30 nm fibers. Such tight coiling is quite often due to repeat sequence DNA.

LAMPBRUSH CHROMOSOMES

Lampbrush chromosomes were first observed in Frog oocytes. Lampbrush chromosomes occur when large amounts of DNA on a normal chromosome (not polytene) are unwound to the 10 nm fiber and RNA is transcribed in large amounts. The loops of 10 nm fiber are observable under a light microscope because they are covered with the RNA that is being transcribed (see figure 15.18 on page 41 of the textbook). LOCUS CONTROL REGIONS, which are sites on chromosomes that appear to regulate the looping in and out of the loops of DNA observed in lampbrush chromosomes. The loops of DNA activated by a specific set of locus control regions are called CHROMOSOME DOMAINS, that each consist of about 50 to 100 kb of DNA, about the same size as the loops on the protein scaffold in bacterial chromosomes..

CHROMOSOME BANDING

We have looked at chromosome banding extensively in the lab. As stated above, the cause of dark G bands is the coiling of the 30 nm fiber; with the light G bands being areas where the 30 nm fiber is not so coiled. G bands are produced by staining chromosomes with a complex of stains called GIEMSA STAIN, which stains the phosphate groups of DNA molecules. To observe bands, chromosomes must be stained and then destained. Areas with the highest concentration of DNA will retain the most stain following destaining, producing bands on chromosomes. Figure 15.19 on page 416 of the textbook shows bands and interbands. Bands themselves contain sub-bands that have been resolved and are used in descriptions of chromosomes. The model shown in figure 15.19 indicates that bands are caused by a folding of the 30nm fiber, with each discrete region of folding called a CHROMOMERE. Sometimes chromomeres are clustered, and a cluster of several small bands will look like one larger band in a crude preparation of G banded chromosomes.

C bands are produced by treating Giemsa stained chromosomes with sodium hydroxide. Only places where DNA is at the highest concentration apparently retains the stain. Such areas of highest DNA concentration appear at the centromeres.

R bands are produced from stained and destained Giemsa preparations, but are viewed using phase contrast microscopy such that light areas appear dark and dark areas appear light. The "R" is for "reverse", and R bands are in fact the lightly staining regions of a chromosome.

We have not fully discussed SATELLITE DNA. Satellite DNA is isolated using density gradient centrifugation, which separates molecules based on their buoyant density in a CsCl gradient. The majority of the DNA of the cell is found at one specific density, but there are other points in the gradient where you find DNA, with such other DNA called satellite DNA. Figure 15.21 on page 417 of the textbook shows a graph of buoyant density vs. amount of DNA as measured by optical density. The 1,700 peak is the main body of DNA. The others are satellite DNA. Satellite DNA is usually composed of repeat sequence DNA that is low in G/C content (the higher the C/G content, the higher the buoyant density). Satellite DNA is found mainly around centromeres, and consists of numerous repetitions of the same sequence.

There are two very obvious structures to chromosomes, the ends, called TELOMERES, and constrictions to which microtubules attach during the M phase of the cell cycle, CENTROMERES.

Centromeres have a protein/RNA structure called a KINETOCHORE associated with them. The kinetochore is the point of attachment of microtubules and associated motor proteins. Most of our basic understanding of centromeres comes from the study of yeasts. The centromeres of two types of yeast are fairly well known, and they are quite different. Bakers yeast (Saccharomyces cerevisiae), consists of 3 basic regions (I, II and III) comprising 125 bp. This centromere interacts with a single microtubules. Schizosaccharomyces pombe is another type of yeast very different from baker's yeast, and has numerous repeat elements (satellite DNA) associated with a central core. The overall centromere consists of about 50 to 100 kb, with the central core making up about 7000bp (7 kb). Other eukaryotes have centromeres that are usually large (i.e. 420 kb in Drosophila melanogaster), but different from any yeast centromere. In figure 15.24 of our textbook, the central core of the Baker's yeast centromere is shown to consist of a modified nucleosome that forms a 20nm POINT CENTROMERE, or CENTROMERE CORE. A microtubule is about 20nm in diameter, and a microtubules is thought to attach at the centromere core.

Telomeres consist of a series of repeats (the sequence AGGGTT..same as TTAGGG...is the basic repeat in human telomeres). As we shall see in the section on DNA replication, telomeres serve to protect the linear chromosomal DNA from degradation during DNA synthesis (see figure 15.27 on page 421 of the textbook). These repeats can form knobs at the end of telocentric chromosomes, and those knobs were called satellites. Telomeres are repeats, low on G/C content, and telomere DNA does not migrate with the main chromosomal DNA in a CsCl density gradient, and thus are satellite DNA. DNA in telomeres loops back on itself to form a circle that protects the ends of chromosomes (see figures 15.29 and 15.30 on page 422 of the textbook) A protein called TRF2 (telomere repeat binding factor) is required for the formation of the loops at the ends of chromosomes. The loop is formed from G-tetraplexes (figure 15.29. See also figure 15.32 on page 423 of the textbook).

One problem that concerned geneticists is called the C-VALUE PARADOX. C-value is the measure of the amount of DNA in a cell. Humans have 3.3 billion base pairs of DNA in each cell. Amoebas have 200 billion base pairs of DNA in every cell. Bony fish have 300 billion base pairs of DNA in every cell. However, less than 500 million base pairs of DNA will account for all the genes in a bony fish, so why does it have 600 times more DNA than it needs? Investigations of the C-value paradox lead to the discovery and characterization of repeat sequence DNA, pseudo genes and junk DNA.

A lot of the DNA in eukaryotes serves no known purpose, and is thus called JUNK DNA. Junk DNA is mostly REPETITIVE DNA, DNA sequences that are repeated many times. Most, but not all, genes are single copy (unique) DNA, which are DNA sequences that occur just once.

Some repetitive DNA consists of copies of functional genes, such as tRNA genes and histone genes (genes that occur in many copies). Some repetitive sequences are copies of functional genes that have been copied and took on different functions. Groups of genes that arise from duplication of some original gene, whether they take on another function or not, are called GENE FAMILIES.

Much of junk DNA are RETROTRANSPOSONS. Retrotransposons are DNA sequences that are copied into RNA. The RNA is then copied into a DNA called COMPLIMENTARY DNA (cDNA), and the cDNA is inserted into the genome. Such DNA elements can occur in hundreds of thousands of copies dispersed throughout the genome. These elements are of two main types, LONG INTERSPERSED ELEMENTS, LINES and SHORT INTERSPERSED ELEMENTS, SINES. LINES consist of thousands of bases, including the genes needed for retrotransposition. About 15% of the human genome consists of LINES.

SINES are shorter sequences, up to a few hundred bases in length, that do not have the genes necessary for retrotransposition, but have none the less been copied into an RNA, which was made into a cDNA that was inserted into the genome by enzymes derived from other sources, like LINES, or retroviruses. Many SINES are derivatives of tRNA genes. One large group of SINES in the human genome are derived from signal recognition particle (srp) genes that are about 300 bases long and occur in about 500,000 copies scattered throughout the human genome. As the restriction endonuclease cuts these sequences, this group of SINES is called the ALU FAMILY, and have been used genomic landmarks, like natural geographic features can be used as landmarks.

Expressed genes that occur in many copies include genes like rRNA genes, genes whose gene products are needed in large amounts for the cell to function properly. The multiples of copies of these genes arose by gene duplication, and in the case of rRNA and histone genes, the copies retain the same function as the ancestral gene sequence. In cases like the globin gene family, copies take on different functions, giving rise to the diversity of globin genes we find among vertebrates (see figure 15.35 on page 425 of the textbook). The globin gene family includes PSEUDOGENES, which are copies of a functional ancestral gene that do not function (usually because initially they lacked a promoter region). Mutations accumulate much more rapidly in in pseudogenes than in functional genes, and thus pseudogenes have been a useful means of assessing differences in mutation rates between functional and non functional DNA.
 

References:

Textbook, pages 407-426

Questions.

4-17, 20, 21, 27 on page 428 of the textbook.