MCB 201 Gene Expression - Spring Semester 2003
Lecture 11 (Morphology and Functional Elements of Eukaryotic Chromosomes cont.)
1. Figure 9-34. Electron micrograph of a histone-depleted metaphase chromosome prepared from HeLa cells by treatment with a mild detegent. Note that this chromosome scaffold, which is primarily protein, has the shape of a metaphase chromosome and persists even after DNase digestion of the DNA loops. Loops containing about one megabase of DNA are attached to the scaffold. These fibers are 30 nm chromatin fibers characteristic of interphase chromosomes.
2. Figure 9-36. Experimental demonstration of chromatin loops in interphase chromosomes. This experiment gives evidence of specific sequences in DNA called scaffold-associated regions (SARs) or matrix-attachment regions (MARs). These sequences bind to the chromosome scaffold. SAR sequences are generally located between transcription units, i.e. genes are located mainly within these chromosome loops. In the experiment shown here, interphase chromosomes were labeled in situ with fluorescent probes consisting of small DNA segments which hybridized to known sequences separated by known distances on cloned, linear DNA. These DNA probes can be distinguished based on fluorescent color differences. The red dots show where the probes bind. Note that some bound probes, known to be separated by millions of nucleotides on linear DNA, are very close together in this chromosome, consistent with the loop structure. The distances measured between bound flourescent DNA probes indicated that loops in the site range from 1 to 4 million base pairs.
3. Figure 9.37. Chromosome domains in nuclei of human interphase lymphocytes. Here is another example of the use of in situ hybridization, this time with biotin-labeled DNA probes specific for sequences along the full length of chromosome 7. Fluorescently labeled avidin, which binds biotin, was used to visualize the positions of the two copies of chromosome 7 in nuclei. Note that the two chromosomes occupy two nonoverlapping regions in the nucleus. This suggests organization of the interphase chromatin into specific domains. Recall that this chromatin is not visible by light microscopy so other experimental approaches are needed to visualize it.
4. Figure 1.9. The Eukaryotic cell cycle. Point out interphase (30 nm fiber chromatin), mitosis (metaphase chromatin) and S phase, when the DNA is replicated.
5. Chromatin contains small amounts of other proteins besides histones and scaffold proteins. These would include transcription factors and replication factors, e.g. polymerases, and a group of proteins called HMG (High Mobility Group) proteins, named for their high electrophoretic mobility compared to other proteins. These proteins appear to be involved in forming and/or stabilizing transcriptional complexes, based on studies with yeast mutants in which normal transcription is altered.
6. Figure 9-38. Banding of metaphase chromosomes helps map the positions of genes and translocations. Chromosomes can be treated in a way that allows for reproducible staining patterns with Giemsa stain for example. The physical and chemical basis for this patterning is unclear, but useful nevertheless as reference points on chromosomes and as ways of identifying individual chromosomes. For example, certain chromosomal translocations are characteristic of specific genetic diseases and specific types of cancer. Here are shown leukemia cells of a particular type of leukemia in which the presence of two abnormal chromosomes are created from a translocation between normal chromosomes 9 and 22. These are the Philadelphia chromosomes (der 22) and an abnormal chromosome 9 (der 9). Panel A shows the detection of these translocations using standard dye banding and Panel B shows the translocations using a technique called fluorescent in situ hybridization or multicolor FISH. These images are created by probes to sequences located throughout the length of the chromosome and these probes are tagged with different fluorescent groups in known proportions, different for each chromosome. Data are collected by a detector mounted on a fluorescent microscope and computer image reconstruction is then used to create false color images of the chromosomes.
7. Figure 9-39. Electron micrograph of a thin section of a bone-marrow
stem cell. Areas of the nucleus that remain darkly staining when the condensed
chromosomes have returned to their decondensed state in interphase are called
heterochromatin. The decondensed, light staining regions are called euchromatin.
Most transcription occurs in the euchromatin regions in the nucleus. Here
we see heterochromatin located near the nucleolus and in association with
the nucleoplasmic face of the nuclear membrane.
What exactly is heterochromatin? The answer seems to be that it is somewhat
heterogeneous. For example, it has been studied in Drosophila polytene chromosomes
where it is often but not exclusively found at the centromeres and telomeres.
Most of the DNA in heterochromatin is simple-sequence or repetitive DNA,
but some genes, probably mostly inactive, are located there as well. (Figure 8-19 from Alberts et al., drawing of polytene
chromosomes of Drosophila).
8. Three functional elements are required for replication and stable inheritance of chromosomes:
9. An example of the use of GFP to study human centromere dynamics - from the GFP In Motion CD.
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