6.2 Chromatin is divided into euchromatin and heterochromatin

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1 6.2 Chromatin is divided into euchromatin and heterochromatin Individual chromosomes can be seen only during mitosis. During interphase, the general mass of chromatin is in the form of euchromatin. Euchromatin is less tightly packed than mitotic chromosomes. Regions of heterochromatin remain densely packed throughout interphase.

2 Heterochromatin May Provide a Defense Mechanism Against Mobile DNA Elements Repeated DNA sequences often are clusters of mobile elements Different elements can replicate through either RNA or DNA intermediates Packaging repeats in heterochromatin may keep them from spreading RNA interference can play a role in silencing and heterchromatin mainenance

3 6.3 Chromosomes have banding patterns Certain staining techniques cause the chromosomes to have the appearance of a series of striations called G-bands. The bands are lower in G C content than the interbands. Genes are concentrated in the G C-rich interbands.

4 Morphology of chromosomes. Satellite Arm Centromere (primary constriction) Secondary constriction Telomere Chromatids

5 The Morphological Chromosome Chromosome appearance varies with stage of the cell cycle and with cell type. Interphase nucleihave distinct regions discernable by staining. Metaphase chromosomes exhibit a condensedstructure and can be distinguished by size and chromosome banding. Polytene chromosomesoccur in insect salivary gland cells. Lampbrush chromosomesare observed during amphibian development. 5

6 Transcriptional Control Transcription controlled by proteins called transcription factors Bind to enhancer DNA Regions of DNA where factors that regulate transcription can also bind Always present in cell, but most likely have to be activated before they will bind to DNA 6

7 Lampbrush Chromosomes Looks like a brush See in most vertebrate oocytes and spermatocytes of some insects = meiotic chromosomes Isolated in diplotene stage of 1 st prophase direct metabolic activities in developing cells they do not condense like the rest of chromosomes initially, later they coil into a normal chromosome

8 Lampbrush Structure Condensed areas in loops along a central axis chromomeres pair in lateral loops adjacent loops present along 1 of 2 axes of chromosome More DNA than to code a single gene Chromosomal loop = 1 strand double helix while central axis is 2 double helices loops are active in RNA transcriptions

9 6.12 Lampbrush chromosomes are extended Sites of gene expression on lampbrush chromosomes show loops that are extended from the chromosomal axis.

10 LampbrushChromosomes and PolyteneChromosomes LampbrushChromosomes Contain Loops of Decondensed Chromatin A remarkable form of chromosome that exists during the extended diplotenephase of meiosis in the growing ovarian eggs of all animals except mammals

11 LampbrushChromosomes DNA loops profusely in Prophase I

12 LampbrushChromosomes Histonesloosen their grip on DNA Transcription proceeds in regions where chromosome packaging has relaxed

13 Although packaged into chromatin, most chromosomes in interphase cells (cells not in mitosis) are too fine and too tangled to be visualized clearly. In a few exceptional cases, however, interphase chromosomes can be seen to have a precisely defined higher-order structure. The meiotically paired chromosomes in growing amphibian oocytes (immature eggs), for example, are highly active in RNA synthesis, and they form unusually stiff and extended chromatin loops that are covered with newly transcribed RNA packed into dense RNA-protein complexes. Because of this coating on the DNA, these socalled lampbrush chromosomes are clearly visible even in the light microscope, where they are seen to be organized into a series of large chromatin loops emanating from a linear chromosomal axis

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15 Lampbrush Chromosomes 15

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17 Lampbrush chromosomes are interphase chromosomes in oocytes with active transcription 20 Stiff extended chromatin loops

18 An RNA processing protein is shown green 21

19 Heterogeneous Ribonucleoproteins Decorate Nascent Transcripts Lampbrush chromosome stained with MAb for hnrnps 19

20 Nucleic acid hybridization experiments show that a given loop always contains the same DNA sequence and that it remains extended in the same manner as the oocytegrows. Other experiments demonstrate that most of the looped DNA is being actively transcribed into RNA. The majority of the chromatin, however, is not in loops but remains highly condensed in the chromomeres, which are generally not transcribed. A general chromosome model based on these studies has been proposed in which loops of 30-nm fibers extend at an angle from the main axis of the chromosome.

21 22 DNA from organisms that usually do not produce lampbrush chromosome is packaged into lampbrush chromosomes-- maybe all are in loop forms but are smaller Actively expressed Not expressed

22 6.13 Polytene chromosomes form bands Polytene chromosomes of Dipterans have a series of bands that can be used as a cytological map.

23 Orderly Domains of InterphaseChromatin Also Can Be Seen in Insect PolyteneChromosomes Because of a specialization (which is different from that of the lampbrushchromosome), chromatin structure is also unusually visible in certain insect cells. Many of the cells of the larvae of flies grow to an enormous size through multiple cycles of DNA synthesis without cell division. The resulting giant cells contain as much as several thousand times the normal DNA complement. Cells with more than the normal DNA complement are said to be polyploid when, as is usually the case, they contain increased numbers of standard chromosomes. In several types of secretorycells of fly larvae, however, all the homologous chromosome copies remain side by side, creating a single giant polytenechromosome. The fact that some large insect cells can undergo a direct polytene-topolyploidconversion demonstrates that these two chromosomal states are closely related and that the basic structure of a polytene chromosome must be similar to that of a normal chromosome.

24 Polytene Chromosomes Found in salivary glands and other tissues of flies and species of protozoans Seen in the nucleus during interphase See linear series of alternating bands and interbands, distinctive banding for each chromosome in a given species

25 Polytene continued Chromomeres individual bands seen in chromosome are lateral condensations of material along the chromosome Polyteneare believed to be: paired homologswhich are usually dispersed as chromatin in somatic cell and not as chromosomes large size and distinctiveness result from many strands of DNA that replicate but do not separate or do cells undergo cytoplasmic division DNA strands in parallel alignment that gives to banding pattern

26 24 Bands (95%) and interbands (5%). Both have genes. Bands do not correlate with genes. Some bands have multiple genes and some lack genes Three times more genes than Bands Different levels of gene expression: Higher at interbands. Heterogeneity of chromosomes (dynamic)

27 Interphase chromosomes in insects-multiple cycles of DNA synthesis without cell division 23

28 Polytene chromosomes are easy to see in the light microscope because they are so large and because the precisely aligned side-to-side adherence of individual chromatin strands greatly elongates the chromosome axis and prevents tangling. Like lampbrush chromosomes, these chromosomes are active in RNA synthesis. Polyteny has been most studied in the salivary gland cells of Drosophila larvae, in which the DNA in each of the four Drosophila chromosomes has been replicated through 10 cycles without separation of the daughter chromosomes, so that 1024 (= 2 10 ) identical strands of chromatin are linedupsidebyside

29 When polytenechromosomes are viewed in the light microscope, distinct alternating dark bandsand light interbands are visible. Each band and interband represents a set of 1024 identical DNA sequences arranged in register. About 85% of the DNA in polytene chromosomes is in bands, and 15% is in interbands. The chromatin in each band stains darkly because it is much more condensed than the chromatin in the interbands. Depending on their size, individual bands are estimated to contain 3000 to 300,000 nucleotide pairs per chromatin strand. Since the bands can be recognized by their different thicknesses and spacings, each one has been given a number to generate a polytenechromosome "map." There are approximately 5000 bands and 5000 interbandsin the total Drosophila genome.

30 6.14 Polytene chromosomes expand at sites of gene expression Bands that are sites of gene expression on polytene chromosomes expand to give puffs.

31 Individual Chromatin Domains Can Unfold and Refold as a Unit Long before anything was known at the molecular level about chromatin structure, studies of polytenechromosomes suggested that a major change in DNA packing accompanies gene transcription, since individual chromosome bands often expand when the genes they contain become active and recondensewhen these genes become quiescent.

32 One of the main factors controlling the activity of genes in polytenechromosomes of Drosophilais the insect steroid hormone ecdysone,the levels of which rise and fall periodically during larval development, inducing the transcription of various genes coding for proteins that the larva requires for each molt and for pupation. As the organism progresses from one developmental stage to another, new puffs arise and old puffs recede, as transcription units are activated and deactivated and different mrnas and proteins are made. From inspection of each puff when it is relatively small and the banding pattern of the chromosome is still discernible, it seems that most puffs arise from the uncoiling of a single chromosome band. Electron microscopy of thin sections of such puffs shows that the DNA in the chromatin is much less condensed than it would be in the 30-nm chromatin fiber. These observations suggest that the chromatin in a band can decondense as a unit during transcription.

33 Puffs Bands are believed to be individual genes, uncoil to undergo transcription and creates a puff know it is transcription because it incorporates a lot of radioactive nucleotides in this area Study of the bands reveal differential gene activity characteristic of pattern of activation during development not sure of the number of genes in each band based on size of band could be ~ average sized genes

34 Chromosome puffs 25

35 RNA synthesis in chromosome puffs 26 New RNA in red Polytene chromosomes from C. tentans Older RNA in blue

36 31 Visualizing transcription (rrna) RNA polymerase