Nuclear Architecture/Overview

• Double-membrane envelope• Has lumen that is continuous with ER• Outer membrane also has ribosomes like ER• Nuclear envelope has pores

– large, complex structures with octahedral geometry

– allow proteins and RNAs to pass– transport of large proteins and RNAs requires

energy• Many nuclear proteins have nuclear localization

signals (NLS)– short basic peptides, not always at N-terminus

Nuclear architecture (cont.)

• nuclear skeleton (lamina)– intermediate filaments (lamins)– anchor DNA and proteins (i.e., chromatin)

to envelope• Nucleolus

– site of pre-rRNA synthesis and ribosome assembly

Tobacco meristem cell : Nucleus with large Nucleolus, and Euchromatin.

Stars indicate heterogeneity in the nucleolus.

Euchromatin

Narcissus flower cell with heterochromatin in the nucleus.

Heterochromatin

d – partially assembled ribosomes passing through pores (side view)

Freeze fracture EM view

c – pores “face on” view thru tunnel

Model of nuclear pore (A is top view)

Fig. 1.37, Buchanan et al.

Nucleolus chromatin spread

RNA Pol I making pre-

rRNAs

Pre-ribosomes

Time-lapse photos of Nucleolus dumping something??

Nuclear Genome in Plants

• DNA organized in chromosomes & replicated as in other systems

• Euchromatin & Heterochromatin (transcrip-tionally inactive) present

• DNA packaged by histones into nucleosomes, then further coiled into 30 nm fibers

• DNA also attached to the nuclear matrix:– SAR (scaffold attachment regions)- A-T rich

sequences that attach DNA to matrix, can promote transcription of “transgenes”

H1 histone

DNA

Nucleosomecore

27Å

110Å

57Å

30 nM Fiber is a Solenoid with 6 nucleosomes per turn

Side view End view

condensation

In Vivo Studies • Promoters of active genes are often

deficient in nucleosomes

SV40 virus minichromosomes with a nucleosome-free zone at its twin

promoters.

Fig. 13.25

Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I.

Packing ratio ~ 25 for this step = 1000 overall

Solenoid attaches to Scaffold, generating Loops

Nuclear DNA also has supercoiled regions.

Fig. 13.14

Genomes & The Tree of Life

• Archaea - small circular genome • Prokarya - small to very small (e.g., Mycobacterium)

circular genomes• Eukarya - 3 genomes

– Mitochondrial – small to micro-sized, linear and circular, prokaryotic origin

– Chloroplast – small, circular, prokaryotic origin

– Nucleus – large, linear chromosomes; evidence of archaea, prokaryotic and “protoeukaryotic?” origins

Plant nuclear genome sizes are large and widely varied.

x 1000 to get bp

Lilium longiflorum (Easter lily) = 90,000 Mb

Fritillaria assyriaca (butterfly) = 124,900 Mb

Protopterus aethiopicus (lungfish) = 139,000 Mb

What about genome complexity?

How many genes do plants have?

Mycoplasma prokaryote 517

E. coli prokaryote 4300

Archaeoglobus archaeon 2500

Cyanidioschyzon rhodophyte 4700

Saccharomyces yeast 6000

Drosophila insect 13,600

Chlamydomonas chlorophyte (unicell)

15,500

Arabidopsis angiosperm, dicot

25,000

Homo sapiens primate 32,000

Oryza (rice) angiosperm, monocot

32-39,000

Organism Taxon # Genes

Texas wild rice

Mycoplasma : How many genes essential for growth (under lab

conditions)?

• Using transposon mutagenesis, ~150 of the 517 genes could be knocked out; ~ 300 genes deemed essential (under lab conditions), which included:– ~100 of unknown function– Genes for glycolysis & ATP synthesis– ABC transporters– Genes for DNA replication, transcription and

translation

Science 286, 2165 (1999)

Features that vary & contribute to the wide range of nuclear genome sizes

1. Amount (or fraction) that is highly repeated

2. Abundance of "Selfish DNA“ (transposons, etc.)

3. Frequency and sizes of introns– Humans have large introns

4. Genetic redundancy

Genetic Redundancy

• The sizes of many gene families have increased much more in certain organisms.

• May account for much of the unexpectedly high genetic complexity of angiosperms

yeast Drosophila Arabidopsis

No. of genes 6200 13,600 25,000

No. of gene families 4380 8065 11,000

No. of genes from duplication

1820 5535 14,000

Genetic Redundancy or Duplication

Impact of Horizontal Transfer on Genomes

• ~ 20% of the E. coli genome was obtained by lateral transfer.

• Not clear how much of plant nuclear genomes are from horizontal transfer– Some pathogens can transfer DNA between

plants– Many nuclear genes came from the prokaryotic

endosymbionts that became Mito. and Chloro.

– Some selfish DNAs such as mobile introns or transposons occasionally transfer horizontally