1. 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

3. 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

4. Tobacco meristem cell : Nucleus with large Nucleolus, and Euchromatin. Stars indicate heterogeneity in the nucleolus. Euchromatin

5. Narcissus flower cell with heterochromatin in the nucleus. Heterochromatin

6. d – partially assembled ribosomes passing through pores (side view) Freeze fracture EM view  c – pores “face on” view thru tunnel

7. Model of nuclear pore (A is top view) Fig. 1.37, Buchanan et al.

8.  Nucleolus chromatin spread RNA Pol I making pre- rRNAs  Pre-ribosomes Time-lapse photos of Nucleolus dumping something??

9. 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”

10. 30 nM Fiber is a Solenoid with 6 nucleosomes per turn Side view End view condensation

11. 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.

12. Packing ratio ~ 25 for this step = 1000 overall Solenoid attaches to Scaffold, generating Loops

13. Nuclear DNA also has supercoiled regions. Fig. 13.14

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

15. 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

16. What about genome complexity? How many genes do plants have?

17. Mycoplasmaprokaryote 517 E. coliprokaryote 4300 Archaeoglobusarchaeon 2500 Cyanidioschyzonrhodophyte 4700 Saccharomycesyeast 6000 Drosophilainsect 13,600 Chlamydomonaschlorophyte (unicell) 15,500 Arabidopsisangiosperm, dicot 25,000 Homo sapiensprimate 32,000 Oryza (rice)angiosperm, monocot 32-39,000 Organism Taxon # Genes Texas wild rice

18. 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)

19. 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

20. 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

21. yeastDrosophilaArabidopsis No. of genes 620013,60025,000 No. of gene families 4380806511,000 No. of genes from duplication 1820553514,000 Genetic Redundancy or Duplication

22. 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