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October 10, 2017

CHROMOSOME STRUCTURE



REFERENCES:
1. Brown TA Genomes, 2nd Ed. Chapter 8
2. Alberts et. al. Molecular Biology of the Cell, Chapter 4
3. Lodish et al. Molecular Cell Biology, Chapter 9
4.Chromosomes and Chromatin, Vol. I-III (1988) Ed. K.W.Adolph. CRC Press.

5. Bodnar, J.W. (1988) A domain model for eukaryotic DNA organization: A molecular basis for cell differentiation and chromosome evolution. J. Theor. Biol. 132:479-507.

Learning checklist:
A. Be able to describe the domain model for chromatin organization in the nucleus.
B. Know what MARs are and their role in gene expression.
C. Understand the experiments which describe the distribution of MARs along the chromosome
D. Understand the evidence that shows the equivalence of scaffold attachment regions (SARs) in mitotic chromosomes and MARs.
E. Be able to list the main differences between heterochromatin and euchromatin.


V.HIGHER-ORDER CHROMATIN STRUCTURE

A. Chromatin in the interphase nucleus is organized into discrete domains defined by sites of attachment to the nuclear matrix.


Chromatin is organized into loop domains by stable attachment to the nuclear matrix at approximately 50,000 base pair intervals. Most domains are condensed into higher order chromatin structures. The DNA of active domains is extended by multiple sequence-specific dynamic associations with the nuclear matrix.



The chromatin is anchored during interphase to the periphery of the nucleus. The protein matrix to which chromatin is anchored is referred to as the nuclear matrix. On the average, attachment to the matrix occurs every 30 to 100 kb. Thus, chromatin is organized into discrete loops, each of which may contain one or a few genes. There are two kinds of matrix, a peripheral matrix, which is primarily on the preiphery of the nucleus, and the fibrilar, or internal matrix, which is primarily in the interior.

There is good evidence that DNA replication and transcription of genes takes place primarily in regions in contact with the internal matrix. In this model, DNA would be threaded through the matrix attachment sites, until the appropriate gene or origin of replication was found. Then replication complexes or transcription complexes would open up the chromatin further, and carry out their functions.

Each domain can be independently regulated. To be transcriptionally active, a domain must be extended (partly uncoiled) into the fibrillar nuclear matrix. Domains that remain coiled are clustered at the periphery of the nucleus. These domains remain transcriptionally inactive. Extended domains are potentially active, but require further developmental or environmental signals to turn on transcription.

EUKARYOTIC DNA SEQUENCE ORGANIZATION IN THE DOMAIN MODEL

Stable attachment sites Dynamic attachment sites
Long range (domain ends) Multiple throughout domain
Stable (covalently or tightly bound proteins) 

= SAR or MAR

Labile (low affinity)
Clustered with: 
  • DNA replication origins
  • enhancers
  • topoisomerase sites
Found everywhere

B. Importance of MARs in gene expression

Breyne, P., Van Montagu, M., Depicker, A. and Gheysen, G. (1992) Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell 4:463-471.

SAR (Scaffold Attachment Regions) = MAR (Matrix Attachment Region)

Tobacco cells were transformed with several constructs:


Undisplayed Graphic

Cells were allowed to develop into callose (undifferentiated) tissue, and assayed using a colorimetric GUS assay (GUS converts the substrate X-gluc into a blue dye). The histogram shows that tissue transformed with either the 35S-GUS gene alone, or 35S-GUS gene plus beta-gobin SAR, vary in GUS activity over a wide range. Notably, pNG6, which has no SARs, has a high percentage of calli with little or no activity. In contrast, calli transformed with GUS + P1-SAR had GUS activities over a more narrow range, with 75% of the transformants falling into a narrow window between 20 and 80 Units of enzyme/mg total protein. What this means is that the presence of MARs flanking a gene appear to create a chromatin domain, leading to a reproduceable level of expression in independent transformants. Without the MARs, expression of a transformed gene is less predictable. Sometimes the gene will be inserted between two compatible MARs, and sometimes it will be inserted in a site that is unfavorable for expression.



 

It is likely that MARs are necessary and sufficient to define chromatin domains. That is, all you need to create a domain is two flanking MARs. Other experiments have shown that a single MAR is inadequate to confer reproducible expression in plants.
 
from Dr. Steve Spiker, North Carolina State University, http://www.cals.ncsu.edu/genetics/spiker/spiker.html

spiker-fig.jpg


C. The distribution of MARs along the chromosome

Arvramova Z, SanMiguel P, Georgieva E and Bennetzen JL (1995) Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1. Plant Cell 7: 1667-1680.

 The underlying organization of chromatin into functional domains can be visualized by identifying MARs on restriction fragments from a large region of the chromosome. The figure at right illustrates one experimental strategy. If the restriction map of a region of the chromosome is known, bands containing MARs can be subtracted from the digest by binding to purified nuclear matrix proteins.

First, a cloned fragment from the region to be studied is digested with one or more restriction enzymes. Next, a matrix protein complex purified from nuclei, is added to the mix. Only DNA fragments containing MARs should be bound by the matrix. Upon centrifugation, the matrix proteins form a pellet at the bottom of the tube, carrying any MAR-containing fragments. The only restriction fragments remaining in the supernatant should be those without MARs. The pellet is resuspended with a detergent to break up DNA/protein complexes, and loaded onto a gel next to a lane containing the original restriction digest.To learn more about the organization of chromatin domains in maize, a 280 kb region flanking the maize alcohol dehydrogenase gene Adh1 was mapped.

The gel at right shows the restriction digestion of the region including fragments 39 through 45, from the map below. Labeled insert DNA was digested with XbaI, XhoI and Bsu36I. 
 
  •  i  the complete set of restriction fragments.
  •  b fragments recovered after binding to nuclear matrix extract.
Image from
Arvramova Z et al. (1995) Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1. Plant Cell 7: 1667-1680.
 



Image from
Arvramova Z et al. (1995) Matrix attachment regions and transcribed sequences within a long chromosomal continuum containing maize Adh1. Plant Cell 7: 1667-1680.

 The map shows the entire 280 kb region assayed for MAR binding activity as shown above. Asterisks (*) indicates restriction fragments which displayed MAR binding activity. The adh1 locus is indicated by an arrow. Fragments hybridizing to RNA transcripts are overlined, indicating the location of genes in this 280 kb region.

What the results tell us:

  1. The size of chromatin domains, as defined by the distances between MARs, can vary from a few kb to tens of kb.
  2. Each domain can contain one or many genes.
  3. Some domains are enriched in highly-repetitive sequences, while others contain mostly single copy sequences.
  4. Domain with repetitive sequences are transcribed.

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Lecture 10, part 1 of 2
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