1. BIOL 200 (Section 921) Lecture # 3, June 21, 2006 Reading for unit 3 on Interphase nucleus (Lecture 3): ECB 2nd edition, Chap 5 pp. 177-191, Chap 15 pp. 502-506, Chap 17 p. 578, Chap 19 pp 651-2. Good questions: 5-1; 5-4 a, b, c; 5-5; 5-7; 5-10; 5-11; 5-12; 5-13; 5-17. Learning Objectives: 1.Understand chromatin structure. Explain how proteins and DNA interact to form chromosomes, starting with the 2nm naked DNA molecule. Be able to explain the structure of nucleosomes and the forces stabilizing these structures, then proceed to the, 10 nm fibre, higher order structures, chromosomal loops, euchromatin and heterochromatin. 2.Understand the organization of chromosomes; describe centromere and telomere. 3.Describe the organization of the interphase nucleus, and the transport of macromolecules into and out of the nucleus

2. Eukaryotic DNA is packaged into chromosomes in plants (Fig. 5-1) and human (Fig. Fig. 5-13) Knobs show ribosomal RNA encoding DNA (rDNA)

3. Replication and segregation of chromosomes during cell cycle [Fig. 5-17]

4. Three DNA sequence elements are needed to produce a eukaryotic chromosome that can be replicated and segregated [Fig. 5-18]

5. The bacterial Nucleoid: (A) A bacterial cell with a distinct nucleoid (B) Release of chromosomal DNA from ruptured bacterial cells [Becker et al. The World of the Cell, 6 th ed.]

6. DNA in mitotic chromosomes is more compact than in interphase chromosomes

7. The interphase nucleus [Fig. 5-19]

8. Two types of chromatin –Euchromatin diffuse in appearance by EM relatively uncondensed contains transcriptionally active genes –Heterochromatin condensed in appearance by EM contains mostly inactive genes constitutive heterochromatin is permanently condensed facultative heterochromatin is specifically inactivated, and depends on the cell type.

9. Beads on String (nucleosomes and spacer DNA) Naked DNA 30 nm Interphase chromatin (packed nucleosomes) Fig. 8-10, p.253 ONLY during cell division, chromatin is further packaged

10. Nucleosomes= DNA+histone proteins Fig. 5-24, p.186 Fig. 5-21, p.184

11. A nucleosome core particle showing DNA tightly wrapped around a histone core

12. 30 nm chromatin fibre Nucleosomes In beads on a string Fig. 5-21, p.184

13. Nucleosomes (basic units of chromatin structure) contain DNA wound around a protein core of eight histone molecules Gel electrophoresis

14. Chromatin remodeling complexes change the nucleosome structure

15. Interphase nucleus

16. Nuclear envelope Is made up of two lipid bilayer membranes, the inner nuclear membrane and the outer nuclear membrane The outer membrane is continuous with the endoplasmic reticulum (ER) membrane The space between the inner and outer membranes is known as the perinuclear space. The nuclear envelope forms a barrier between the cytoplasm and the nucleus and controls the passage through nuclear pores of macromolecules in and out of the nucleus.

17. Macromolecular transport into and out of the Nucleus [Becker et al. The World of the Cell, 6 th ed.]

18. Nuclear transport of macromolecules Macromolecules transported into the nucleus: –DNA replication enzymes, –nuclear lamina proteins, –nuclear matrix proteins, –ribosomal proteins, –chromatin proteins (histones), –transcription factors, –structural proteins. Macromolecules transported out of the nucleus : –messenger RNA, –transfer RNA, –ribosomal subunits.

19. Nuclear pores Are protein complexes that span both nuclear membranes, Regulate the passage of macromolecules between cytoplasm and nucleoplasm Allow movement by diffusion of small molecules Provide for regulated active transport of macromolecules and macromolecular assemblies such as ribosomal subunits. The pore complex is 70-90 nm in diameter and the opening is thought to be about 9 nm in diameter.

20. Nuclear pore complexes allow transport of molecules into and out of the nucleus

21. Transport of proteins through nuclear pores Active transport – requires GTP hydrolysis Prospective nuclear proteins have a nuclear localization signal (NLS): -Pro- Lys-Lys-Lys-Arg- Lys-Val-

22. Intermediate filaments of the nuclear lamina support and strengthen the nuclear envelope [Fig. 17-8]

23. The nuclear envelope breaks down and reforms during mitosis [Fig. 19-18]

24. Nuclear lamina (nuclear cortex) - an example of a non-chromatin protein in the nucleoplasm. The inner surface of the nuclear membrane is asociated with a protein complex called the nuclear lamina. The lamina supplies an internal structure for the nucleus The interphase chromosomes bind to the nuclear lamina. The lamina proteins help the nucleus to reorganize after mitosis The lamina binds other membrane receptors in the inner nuclear membane

25. Interphase Nucleus Heterochromatin Heterochromatin: dark, condensed DNA that is transcriptionally inactive during interphase. Euchromatin Euchromatin: lighter, transcriptionally active DNA Nuclear Envelope Nuclear Envelope: Contains a double membrane surrounding the nucleus. Contains nulear pores throughout. Nucleolus Nucleolus: site of ribosomal assembly and rRNA transcription

26. Nucleolus Is a unique region within the nucleus It consists of chromosomal regions containing the ribosomal RNA transcription units It is the site of ribosomal RNA synthesis It is the site of assembly of ribosomes It consists of a fibrous component (DNA and RNA transcripts) and a granular region consisting of developing ribosomal subunits.

27. Nucleoplasm - (inside nucleus, lumen of the nucleus) Semifluid matrix Made up of extended chromatin (DNA and protein complexes) and non-chromatin proteins (i.e. nuclear matrix, nuclear lamina) Its function is to contain the genetic material and serve as a site for DNA replication and for transcription of the genetic material.