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1 1. 2 CHAPTER 8 NUCLEUS AND CHROMOSOME 3 Nucleus is the most important organelle in cell. In mammalian cells, excepting RBC, all cells else are the.

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Presentation on theme: "1 1. 2 CHAPTER 8 NUCLEUS AND CHROMOSOME 3 Nucleus is the most important organelle in cell. In mammalian cells, excepting RBC, all cells else are the."— Presentation transcript:

1 1 1

2 2 CHAPTER 8 NUCLEUS AND CHROMOSOME

3 3 Nucleus is the most important organelle in cell. In mammalian cells, excepting RBC, all cells else are the nucleus contained cells. In prokaryotic cells, there is no membrane to package the nucleic acid substance, so, we call this nucleic substance enriched area as “Nucleoid”. The major structures of nucleus include: ① nuclear envelope. ② nucleolus. ③ nuclear matrix. ④ chromatin. ⑤ nuclear lamina. The major functions of nucleus: ① inheritance: maintain the genetic continuity of generation by the replication of DNA chromatin and the proliferation of cell. ② development: regulate the cell differentiation by the regulation of spatiotemporal sequence of gene expression.

4 4 Structure of nucleus: (n: nucleolus; N: euchromatin)

5 5 I. Nuclear envelope (Nuclear membrane) Nuclear envelope is the lipid bilayer that packages the nucleus. Nuclear envelope separates the DNA from cell plasma and forms a stable inner environment to: ① protect the DNA from damage, ② separate the replication of DNA from the translation of RNA spatiotemporally, ③ the chromatin is anchored on to the nuclear envelope, that is beneficial to be despiraled, replicated, condensed, and distributed into new nuclei equally, ④ the pores on the envelope are the channels for the substance exchange. Nuclear envelope is bilayer membrane: Nuclear envelope is composed of inner nuclear membrane, outer nuclear membrane, and perinuclear space. There are nuclear pores on the membrane that are linked with plasma. Ribosome is attached to the plasma side of outer nuclear membrane, and the ribosome is linked with ER. The perinuclear space is linked with ER space. The intermediate filament (10nm) is attached to the outer nuclear membrane, so, the locations of nucleus and ER are not movable because of the intermediate filament. The unmovable locations are convenient to the co-function of nucleus and ER. The nuclear lamina (meshwork filament proteins) attached to the inner side of the inner nuclear membrane can stabilize nuclear membrane shape.

6 6 The photo of nuclear envelope by TEM

7 7 Structure of nuclear envelope

8 8 The functions of nuclear lamina: 1 . Keeps nuclear shape no changed: If you use the high concentration salt solution, detergent or nuclease to move away the nuclear substance, the remaining (nuclear lamina) still presents a nuclear shape. In addition, the nuclear lamina links nuclear skeleton meshwork and intermediate filament together to form a continued meshwork for nucleus. 2 . Is associated with the assembly of chromatin and nucleus: The shape of nuclear lamina can be changed during the cell proliferation phases. In the G1 phase, nuclear lamina can present the anchoring sites for heterochromatin on the inner side of inner nuclear membrane. At the ending of the G1 phase, the nuclear lamina will be phosphorylated and the nuclear envelope will disappear. The B type of nuclear lamina will combine to the residual vesicles of nuclear membrane, and A type of lamina will be dissolved in plasma. In the later of M phase, All types of lamina will be dephosphorylated and assembled again to form nuclear lamina and mediate the nuclear envelope construction.

9 9 Structure of nuclear lamina

10 10 The nuclear pores are the channels for the substance transportation: Nuclear proteins are synthesized in plasma, then will be imported into nucleus by the pores. The RNAs and the ribosome subunits synthesized in nucleus will be exported into plasma by the pores also. In addition, it is indicated by a injection experiment that small molecules can enter the nucleus by diffusion from the pores. Nuclear pores are composed of 50 different nucleoporins at least, and we call these pore structure as nuclear pore complex (NPC). Usually, a mammalian nucleus contains 3,000 nuclear pores. The more activities a cell takes, the more nuclear pores the cell contains. For example, a frog ovum can contain 37.7X10 6 nuclear pores, but a matured cell contains 150~300 nuclear pores only. The structures of nuclear pore include ① cytoplasmic ring located on the cell plasma part of the pore complex contains 8 filaments extending into plasma. ② nuclear ring located on the nuclear plasma part of the pore complex extending 8 filaments also. ③ transporter located in center of the pore as a plug particle. ④ Spoke located on the edge of the pore as the spines.

11 11 The nuclear pore structures on the cell plasma side after an extraction

12 12 The nuclear pore structures on the nuclear plasma side after an extraction

13 13 A model structure of the nuclear pore

14 14 The transportation by nuclear pore is associated with signal transduction: 1982, R. Laskey identified a signal sequence on the C terminal of nucleoplasmin enriched in nucleus, and the signal can lead protein to enter nucleus. This signal sequence was named as nuclear localization signal (NLS). The firstly identified NLS is the T antigen of SV40. This antigen is synthesized in cell plasma, and transported into nucleus quickly. Its NLS is pro-pro-lys- lys-lys-Arg-Lys-val. NLS is composed of 4 – 8 amino acids containing Pro, Lys and Arg. NLS is not specific to target protein and will not be cleaved by protease. Karyopherin is a protein family that is associated with the selective transportation by the pore, and it is a receptor family actually. The imporin of them imports proteins into nucleus from cell plasma and the exportin of them exports the proteins on an opposite direction. Ran is another protein involved in the transportation by the pore complex. Ran is a G protein that regulates the assembly and disassembly of the complex of the protein transported and the receptor used. Ran-GTP concentration is much higher in the nucleus than in cell plasma.

15 15 Gold labeled nucleoplasmin is passing through the nuclear pore

16 16 Nuclear plasma protein (nucleoplasmin) is transported by the following steps: ① The protein combines to the α / β dimer of the receptor (imporin). ② The complex of the protein transported and the receptor used combines to the filaments located on the NPC cytoplasmic ring. ③ The filaments curve to the nuclear center, the transporter structure will be changed to form a hydrophilic channel, and the protein passes through the channel. ④ The complex of the protein transported and the receptor used combines to Ran-GTP, the complex is disassembled and releases out the protein transported. ⑤ The imporin β combined with Ran-GTP will be exported out of the nucleus, the GTP combined with Ran will be hydrolyzed in cell plasma, and the Ran-GDP will go back to nucleus to be transformed to Ran-GTP again. ⑥ The imporin α will be transported back to cell plasma with the help from exportin. We know a little about how the macromolecules are transported to cell plasma from nucleus. In most of cases, the RNA in nucleus is combined with protein to form an RNP complex, then, transported into cell plasma. There is nuclear exportation signal (NES) on the protein of RNP complex that can combine to the intracellular receptor, exportin, to form the complex of RNP- exportin-Ran-GTP. In the cell plasma, this complex will be disassembled and release out the Ran-GTP, RNA, Ran-GDP, exportin, and RNP protein.

17 17 The nucleoplasmin is transported into nucleus

18 18 II. Chromosome Chromatin was named by W. Flemming in Chromosome was named by Waldeyer in Chromatin and chromosome are same substance with different shape presentation in different cell cycle phases. The chemical components of chromatin: Chromatin is composed of DNA, histone, nonhistone protein, and some RNA at ratio about 1:1:(1-1.5):0.05. DNA: DNA is the carrier of genetic information. DNA sequences can be sorted as 3 types: nonrepeated fraction, moderately repeated fraction ( ), and highly repeated fraction (>105). DNA forms: B-DNA, Z-DNA, and A-DNA.

19 19 DNA forms (Red color shows the couple backbones)

20 20 Chromosome DNA contains three basic sequences: ① autonomously replicating DNA sequence (ARS). ARS is the starting site of DNA replication. In yeast genome, there are ARSs included, and most of them contain a AT enriched 11bp sequence called as ARS consensus sequence (ACS). ② centromere DNA sequence (CEN) composed of a lot of repeated sequences. ③ telomere DNA sequence (TEL). TEL is similar in different bio organisms, and composed of 5 – 10bp repeated sequences. Human TEL repeated sequence is TTAGGG. In 1983, A. W. Murray et al constructed yeast artificial chromosome (YAC) contains ARS, CEN, TEL and exogenous DNA with the length of 55kb. YAC is very useful to transgenic technology and construction of cDNA library because the length of insert to YAC can be much longer than that to plasmid.

21 21 Three basic sequences of chromosome

22 22 Histone: Histone is positively charged and contains arginine and lycine. Histone is alkaline protein. Histones can be sorted as two types: 1. Highly conserved core histone including H2A, H2B, H3, and H4. 2. Non conserved linker histone including H1 only. The core histone is highly conserved, especially the H4 is. For example, 2 of 102 amino acids of the H4 of cattle and pea are different, but cattle has been evoluted 300 million years earlier than pea. The reasons for that may be as the follows: 1. Most of the amino acids of core histone interact with DNA or other histones, so, any change of them will cause the fatal mutation. 2. In all bio organisms, the DNA phosphodiester skeleton that interacts with histone is same. The core histone head part makes DNA winded round the histone center by the electronic force between arginine residue and phosphodiester skeleton. By the described as above, nucleosome can be formed. The tail part of core histone containing a lot of arginine and lysine residues. The tail part is the site to be modified after translation. H1 is easy to be mutated, and it is species specific and tissue specific.

23 23 Nonhistone protein: Nonhistone protein is the protein that binds to the specific DNA sequence of chromosome, so, we call it as sequence specific DNA binding protein. The features of nonhistone protein are as the follows: ① Nonhistone protein is negatively charged and acidic protein that contains a large number of aspartic acids and glutamic acids. ② Nonhistone protein can be synthesized during the whole cell cycle, but histone protein is synthesized during the S phase only. ③ Nonhistone protein can recognize the specific DNA sequence. The functions of nonhistone are as the follows: ① Help DNA molecules to be pleated and form different structure domains that are beneficial to DNA replication and gene transcription. ② Help to start DNA replication reaction. ③ Regulate transcription and gene expression.

24 24 From DNA to chromosome: There are 23 pairs of chromosomes in a human nucleus. If you open and extend the DNA molecule in each chromosome, it will be 5cm long. If you link all DNA molecules in a nucleus together, it will be 1.7 – 2.0 m long. But, the diameter of nucleus is shorter than 10μm. That is why I told you the genome information is packaged into the space of a cell nucleus —— thousands of times smaller than the dot on this i. The primary structure formed by the powerful compaction is called as nucleosome. Nucleosome: If the chromatin is treated by a nonspecific nuclease, the DNA fragments around 200bp can be obtained in most of cases. If you treat the null DNA with that enzyme, you will obtain the randomly degenerated fragments of DNA. Based on this experiment, R. Kornberg figured out the model of nucleosome. Nucleosome is a beaded structure composed of core particles and linker DNA. We can describe the structure as the follows: ① Each nucleosome includes about 200bp DNA, one histone core, and an H1. ② The octameric histone core is composed of 8 molecules from H2A, H2B, H3, and H4 by two molecules from each. ③ DNA molecule winds the core particle with a left hand helix and 80bp for each circle circles for each structure. ④ Adjacent core particles are linked by a 60bp linker DNA.

25 25 Structures of nucleosome

26 26 Chromatin DNA filament: The DNA is compacted to be shortened by 7 folds and forms the DNA filament in 11nm diameter when it was transformed to the beaded nucleosome chain. Chromatin DNA exists in another style by that the beaded nucleosome chain is condensed by 6 folds. Under electron microscope, we can see the chromatin DNA filament in 30nm diameter that is formed by the overlapped helix structure of the beaded nucleosome chain.

27 27 The DNA filaments in 30nm and 11nm diameter (A) Is composed of (B)

28 28 For the advanced package of the chromosome, we keep detail unknown so far. Probably, it is the serial overlapped or pleated like the follows: From DNA to Chromosome: DNA 11nm filament (beaded nucleosome chain) 30nm filament pleat as loop chain bind to the sites on nuclear skeleton where is AT enriched assembly of chromosome

29 29 Assembly of chromosome

30 30 Heterochromatin and euchromatin: In the inter phase (G1 and G2) of cell cycle, the chromatin in the nucleus can be sorted as heterochromatin and euchromatin. Euchromatin is the DNA regions where the transcription is very active. Euchromatin looks like loose loop and bright staining under electron microscope. Euchromatin is easy to be cleaved by nuclease at some hypersensitive sites. Heterochromatin is condensed in G phase without any transcription, so, it was named as inactive chromatin. Heterochromatin is the genetic lazy regions, and replicated lately, condensed early, that is called as heteropyknosis.

31 31 Heterochromatin (dark staining) and euchromatin (bright staining) Heterochromatin Euchromatin

32 32 Constitutive hetero- chromatin is hetero- pyknosed chromatin in each type of cell and located in centromere region. The Fig shows you the Constitutive hetero- chromatin displayed by fluorescence hybridization in situ.

33 33 Facultative heterochromatin is heterochromatin appeared in some special cell type or developing stage. The X chromosome of female mammalians is the facultative heterochromatin. Usually, female mammalian cell contains double X chromosomes, and one of them is heterochromatin called barr body. When a human embryo is developed after 16 days, one X chromosome will be transformed as barr body with dark staining. So, we can identify the sex of a human embryo by checking the barr body of the embryo cells in the amniotic fluid. The barr body like a drumstick in a white cell

34 34 The structure of chromosome: In the M phase of cell cycle, chromatin will be transformed as chromosomes by the powerful condensation. Chromosomes are stick shape with different length. The metaphase chromosome is the best stage to observe and number them because the morphology of chromosome is stable at this time. The number of chromosome is same in the same type of cells from different individuals of one species. The chromosomes of sex cells are haploid, we mark it as n. The chromosomes of other cells are diploid, we mark it as 2n. The chromosomes of some cells of some species are polyploid, such as, 4n, 6n, and 8n. The different cells from same individual can be different chromosome types. For example, body cells of rat are 2n, but its liver cells can be 4n, 8n, and 16n. The chromosome number of human endometrial cell is variable from 2n =17 - 2n =103, that is not euploidy. The chromosome number can be different in different species cells. For examples, human 2n = 46, chimpanzee 2n = 48, fruit fly 2n = 8, wheat 2n = 42, rice 2n = 24, onion 2n=16.

35 35 The terms used to the structure of chromosome: 1 . Chromatid: Metaphase chromosome is composed of two chromatids with a junction at the centromere site. Each chromatid is formed by the overlapped and pleated DNA double strands. When the cell is dividing the chromatids can be separated into two new cells. 2 . Chromonema: In the S or G phase cells, each chromonema indicates a chromatid. 3 . Chromomere: Chromomere is the linear beaded particles chain DNA. The chromomere of heterochromatin is bigger than that of euchromatin. 4 . Primary constriction: It is a bright stained hang ditch on the metaphase chromosome where the centromere is located, so, it can be called as centromere region. Each chromosome has one localized centromere. The chromosome from some species has centromere function every where. We call this chromosome as holocentromere chromosome. For examples, ascarid (round worm) and other nemas, butterfly. The chromosomes can be sorted by the location of centromere as following: ① metacentric chromosome. ② submetacentric chromosome. ③ subtelocentric chromosome. ④ telocentric chromosome. 5 . Secondary constriction: Excepting primary constriction, the second ditch is called as secondary constriction. The location of secondary constriction is unmovable by that we can identify chromosome.

36 36 The terms of chromosome structure

37 37 6 . Nucleolar organizing regions (NORs): They are the areas where the genes for ribosome RNA are located. They can synthesize the 28S, 18S, and 5.8S rRNA for ribosome. NORs can exist in secondary constriction. 7 . Satellite: It is a ball part located at the terminal of chromosome, and linked to the main part of chromosome by secondary constriction. The satellite located at terminal of chromosome is called as terminal satellite, and located between two secondary constrictions is called as intermediate satellite. 8 . Telomere: It is the specialized part located at the terminal of chromosome. The function of telomere is maintenance of the stability of chromosome. Telomere is composed of the highly repeated fractions, and it is so conserved that it is similar between the totally different life beings. The component of human telomere is TTAGGG. Telomere is associated with aging. After each replication of telomere DNA, the telomere will be shortened by 50 – 100bp. The replication of telomere is droved by telomerase that has reverse transcriptase activity. This enzyme lacks in normal cells, so, telomere will become short with the cell proliferation. So, cell will be aging during this action.

38 38 The nucleolus will be formed in the center of nucleolar organizing regions

39 39 The telomeres displayed using fluorescence hybridization in situ. The sequences of telomere

40 40 The structure of centromere: Centromere and kinetochore are different concepts, the former means the special region by that the chromatids of metaphase chromosome are linked together, and the later means the outer surface structure located on the primary constriction that is linked to spindle fibers. Centromere contains 3 domains: kinetochore domain, central domain, paring domain. Three domains of centromere

41 41 Kinetochore domain: Kinetochore domain is composed of outer plate, inner plate, interzone, and fibrous corona. The inner plate combines to the heterochromatin of central domain, outer plate combines to filaments of spindle fibers. Their motor proteins located on the fibrous corona to supply energy to the chromosomes separation.

42 42 Central domain: It is located below the centromere and contains the heterochromatin composed of highly repeated α satellite DNA. Paring domain: It is located in centromere by that the chromatids of metaphase chromosome are linked together. There are two types of proteins in this domain: inner centromere protein (INCENP), and chromatid linking protein (CLIP). By using anti-centromere antibodies (ACA), INCENP or CENP (centromere protein) can be identified and sorted as the follows: TypesFunctions CENP-A Specific histone to centromere CENP-B Binds to satellite DNA in central domain CENP-C Binds to kinetochore CENP-D CENP-E Drive motor protein CENP-F Binds to kinetochore INCENP-A Link partner chromatid INCENP-B

43 43 Karyotype and bands display: The bands display technology of chromosome was developed in 1960s to 1970s, and it brought the chromosome researches to a new and fast developing stage. The result data about chromosome bands is the very useful background research for modern genome research programs, gene molecular research programs, and genetic research programs. Karyotype is the total features of the chromosomes in M phase. It includes the number, size, and shape of chromosome. If the paired chromosomes are arranged by shape and size, a figure will be obtained, and we call it as karyogram. Karyogram is of characters of species.

44 44 Karyogram of Platypleura kaempferi

45 45 The chromosome banding technology is very important to genetics research, species classification, and others. This technology includes the cell and chromosome treatments by physical and chemical methods, chromosome staining and bands display. Bands display technologies can be sorted as two types: 1. The bands distribute on entire chromosome, such as G, Q, and R banding technologies. 2. The bands distribute in localized region of chromosome, such as C, Cd, T, and N banding technologies.

46 46 Human G Karyotype. G bands display the regions where AT is enriched

47 47 Human Q Karyotype. Q bands display same thing of G bands

48 48 Human C Karyotype. C bands display the centromere heterochromatin

49 49 Special chromosomes: Polytene chromosome: It was identified in some insect saliva cells in Polytene chromosome: ① 1,000 – 2,000 folds huger than others. It is the reason that chromosomes are replicated without separation. ② Polytene. Each polytene chromosome is composed by 500 – 4,000 helix opened chromosomes. ③ Cell junction and homologous chromosomes combination. ④ Striation. ⑤ In some life stage of insects, some bands of polytene chromosome become loosed and form puff and Balbiani ring. Puff can be labeled by H3-TdR, that means puff is the region where the gene transcription is very active.

50 50 The chromosomes from saliva cells

51 51 Lamp-brush chromosome: Lamp-brush chromosome was identified in fishes firstly. There are lateral loops on the chromosome like lamp brush. It is composed of two homologous chromosomes. Lateral loops are the region with RNA active transcription. B chromosome: In 1928, Randolph, a scientist, call normal chromosomes as A chromosomes, and call abnormal chromosomes existed in many animals and plants as B chromosome.

52 52 III. Nucleolus Nucleolus may be visible in G phase nucleus. They are spherical and 1 – 2 for each cell usually. The number and size of nucleolus are depended on the cell type and function. The more proteins synthesis and the faster proliferation the cell takes, the more and bigger nucleoli the cell has. Nucleolus disappears before the cell division, and appears in the end of division. The major functions of nucleolus are rRNA transcription and ribosome assembly. Structure of nucleolus: No any membrane packages nucleolus area. There are three special areas can be identified under electron microscope: ① fibrillar centers (FC) that are surrounded by dense fibers, and low electric density. FC contains RNA polymerase and rDNA that is naked molecule. ② dense fibrillar component (DFC) that is a loop or half loop to surround FC. Transcription is carried out in the border region of FC and DFC. ③ granular component (GC) composed of 15-20nm particles that are the RNPs in different manufactured steps. RNP means the RNA combined with protein. Nucleolus chromatins can be sorted as two types: heterochromatin and euchromatin. The nucleolus heterochromatin is always located around the nucleolus, so we call them as nucleolus peripheral chromatin. The nucleolus euchromatin is located in nucleolus, and nucleolus organizing region in that the rDNA is located.

53 53 Structure of nucleolus

54 54 IV. Ribosome Ribosome is the manufacturing shop to synthesize proteins. There are about 20,000 ribosomes in an actively growing bacterium. Ribosome proteins are 10% of total proteins of cell, and its RNA is 80% of cell total RNA. Structure of ribosome: The ratios of protein and RNA to ribosome components are 40% and 60%. The ribosome subunits are composed of the combination of the protein and RNA. The catalytic activities needed by the translation are presented by ribosome protein, rRNA and other helper factors. The ribosomes can be sorted as two types. 70S ribosome exists in bacteria, mitochondrion, and chloroplast. 80S ribosome exists in the plasma of eukaryotic cells. Ribosome is composed of a large subunit and a small subunit. The both subunits will be combined together when the ribosome synthesizes protein with mRNA as template. After the translation, the ribosome will be separated as two parts again. When a protein is translated on an mRNA, many ribosomes can bind to the mRNA to synthesize the protein. We call these ribosomes for one protein synthesis as polyribosome. The longer mRNA is used, the more ribosomes are combined. The polyribosome enhances the efficiency of protein synthesis. Prokaryotic 5S rRNA and eukaryotic 5.8S rRNA are very conserved for their structures, so, they can be used to research the bio-evolution.

55 55 Assembly of ribosome: The DNA fragment encoding rRNA is called as rRNA gene. There are about 200 copies of this gene in a human cell. rDNA contains no histone core, so, it is a naked DNA. To transcript rRNA, the RNA polymerase moves ahead along the DNA molecule. The synthesized rRNA molecules extend out their molecules from the complex of polymerase and DNA, and form a featherlike structure under microscope.

56 56 rRNA transcription The filaments are the new synthesized 45S rRNA that combines to protein to form RNP complex. The methylated 45S rRNA can be cleaved as the two parts by RNase: 18S rRNA and 32S rRNA , the latter is cleaved as 28S rRNA and 5.8S rRNA. The synthesized 5S rRNA will be transported into nucleolus to join the assembly of the large subunits of ribosome. There is a 60bp non-transcription DNA fragment between adjacent rRNA genes

57 57 Assembly of ribosome

58 58 Model of a ribosome mRNA Synthesized protein

59 59 V. Nuclear matrix Nuclear matrix is called as nucleoskeleton that is a meshwork in eukaryotic cells, that is what I told you before. Because nuclear matrix is associated with DNA replication, RNA transcription and modification, chromosome assembly, and virus replication, nuclear matrix is now paid more attentions to. Components of nuclear matrix: ① Non-histone filaments at ratio of 96%. The nucleoskeleton contains three scaffold proteins: SC Ⅰ, SC Ⅱ, and SC Ⅲ. ② A little RNA and DNA: The RNA is important to maintain the skeleton structure. The DNA is called as matrix /scaffold associated region (MAR or SAR) where the AT is enriched to form the heterochromatin binding sites. ③ A little phospholipids (1.6%) and sugars (0.9%). Nuclear skeleton – nuclear lamina – inter filaments – pore complex is a meshwork system with very good stability.

60 60 The function of nuclear skeleton: 1.Present the scaffolds for DNA replication. DNA can be anchored on to the scaffold with a replication loop. The enzymes needed by DNA replication are located on the skeleton, such as DNA polymerase α, DNA primerase, DNA topoisomerase II. 2. Is the place where gene can be transcripted and modified. There are RNA polymerase binding sites on the skeleton. New synthesized RNA is combined to the skeleton for further modification. 3. Is associated with the assembly of chromosome. The nuclear skeleton may be same thing to chromosome skeleton. 30nm chromatin fibers are combined to nuclear skeleton to form loops that will be packaged further in M phase to be assembled as chromosome.

61 61 Chromatin bound on nuclear skeleton or chromosome skeleton


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