Nuclear Structure and Function Research Group

Objectives
The material covered in this lecture, coupled with the recommended reading, should enable you to:
• appreciate the size of nuclei and the different structures in it
• describe the different structures (types of chromatin, membrane, pores, nucleolus)
• understand how long DNA molecules are packed into nuclei
• describe the crucial nuclear functions (replication, transcription, repair, recombination)
Overview
Most conspicuous organelle in cell; diameter 2-10 micrometer (Fig); arguably most important (contains genome).
Function: contain/protect genome, carry out replication, transcription, repair, recombination, ribosome production.
Genes surrounded by envelope of 2 membranes.
Nuclear pores pierce envelope and allow communication between nucleus and cytoplasm.
Nuclei generally have little internal structure visible in the light microscope, apart from dense nucleoli (sites where rRNA is synthesized, ribosomes assembled).
Most of rest of nucleus filled with chromatin, recognized by reaction with cationic (basic) stains (eg methylene blue, hematoxylin). Cellular components that bind basic and acidic dyes are termed basophilic and acidophilic.
Chromatin commonly divided into 2 types: euchromatin (dispersed appearance, occupies most nuclear volume), heterochromatin (densely packed with fibres, structure more like that in mitotic chromosomes; Fig). Heterochromatin often condensed against envelope or nucleolus, can aggregate into densely-staining - often internal - chromocentres. 2 kinds of heterochromatin: constitutive (never expressed, often contains short repeated DNA sequences) and facultative (expressed in some cell lineages). Excellent example of latter is X chromosome of mammals. In female cells, one X (selected randomly early during development) is heterochromatic, (almost entirely) transcriptionally inactive, forms dense Barr body under nuclear membrane; other (with essentially same DNA sequence) is euchromatic, transcriptionally active.
Size and structure
Typical nucleus of animal cell is ~10 µm in diameter; occupies only a small fraction of the cell (eg 6% vol of a mammalian liver cell, 22% is mitochondrial).
Nuclei containing different amounts DNA may have roughly the same size, and - conversely - those with same amount DNA can have very different sizes (eg nucleus in resting human lymphocyte is ~5 µm in diameter, that of migratory neuroblast can be >20 µm).
DNA in a human chromosome is arguably the longest and most important biomolecule (Fig)
Widely assumed there are ~3,000,000,000 bp (or 3,000 Mbp) in a haploid human nucleus.
Typical human chromosome contains ~100 Mbp DNA - 2 nm wide and 3.4 cm long when stretched out; 3,000 Mbp DNA would cover 2 m. [Values obtained by multiplication of number of bp by distance between two in Watson-Crick structure (ie ~0.34 nm).]
Difficult to grasp dimensions of molecules with such length-to-width ratios because they are so far outside anything found in everyday world. An analogy: a kite string with ~2 mm dia is a millionfold wider than DNA fibre - if it had same length-to-width ratio as 100 Mbp of DNA, it would be 30 km long! String equivalent to 6000 Mbp in diploid nucleus would be 2000 km (connects London-Rome).
Packing problem - reduce contour length by ~10,000x.
So tight packing - easy to imagine tightness of packing controls access of polymerases to DNA, and so gene activity.
DNA is strong acid - must be neutralized - histones H1, H2A and B, 3, 4 (contain high proportion of positively charged amino acids - lys, arg).
Histones highly conserved (2 differences in aa sequence of H4 from peas, cows).
Non-histone chromatin proteins (many bind to specific DNA sequences).
DNA coiled around nucleosome, into solenoid (?), loops, chromosome domains/territories (Fig).
Nuclear envelope - distinguishes eu- from pro-karyotes (Fig).
Double membrane of 2 lipid bilayers (inner, outer nuclear membranes) separated by 20-40 nm (perinuclear space). In EM, outer membrane continuous with membrane of endoplasmic reticulum (ER); often outer surface studded with ribosomes like cytoplasmic face of rough ER. Space between nuclear membranes continuous with lumen of ER, so outer membrane as specialized region of ER.
A nuclear lamina - largely responsible for determining nuclear shape - underlies nucleoplasmic side of inner membrane. Made of fibrous mesh of lamin proteins, members of intermediate filament family (family contains keratins, vimentin).
Nuclear pores - each surrounded by a nuclear pore complex - pierce membrane (Fig, Fig).
Pore is gate allowing selective transport in and out.
Each pore enormous (~125 x 10⁶ D; ~100 different polypeptides). 8 large protein granules arranged in a circle around central hole (internal and external diameters of ~80 and ~120 nm). 8 fibrils attached to cytoplasmic side of the ring, 'basket' on other side.
Central hole behaves like aqueous channel of ~9 nm that allows passive diffusion through the membrane (proteins of >9 nm cannot diffuse in/out).
Traffic density very high - in human cells ~50 histone molecules and ~100 ribosomal proteins enter every minute through each pore, while ~2 ribosomal subunits (dia ~15 nm) exit!
Functions - gene transcription, replication, repair, recombination.
Label newly-made RNA and DNA with appropriate precursors (eg uridine and UTP/thymidine and TTP, tagged with ³H, ³²P, Br, biotin) to label nascent RNA/DNA. Note phosphorylated precursors cannot cross cell membrane.
Active polymerases concentrated in discrete transcription/replication 'foci' or 'factories' (Fig).
28, 18, 5S rRNA made in nucleolus, a ribosome-producing factory (Fig). The nucleolus is the most prominent cytological feature of nucleus, with high concn RNA but little DNA. Its size reflects activity - small in dormant cells, can swell to 25% nuclear vol in cells actively making protein.
Reference: Chapter 4 in Alberts, B. et al. (2002). 'Molecular Biology of the Cell'. 4th Edition. Garland Publishing, NY and London.
Chapters 1 and 2 in Cook, P.R. (2001). 'Principles of Nuclear Structure and Function'. J. Wiley and Sons, New York. [Web link]
Pollard, T.D. & Earnshaw, W.C. (2004). 'Cell Biology'. WB Saunders/Elsevier.

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