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PLNT 3140 Introductory Cytogenetics - 2022

Intrinsic Features of Chromosomes

Learning Objectives

Read before class: How DNA replication works

While our task of building a chromosome has been of enormous scope, the main thing we've done so far is simply to describe how DNA is packaged in chromosomes, and some of the functional ramifications of that packaging. We still need to identify some important functional features of chromosomes that make it possible for them to replicate and segregate. We will describe a minimal set of features required for normal replication and segregation. These are centromeres, origins of replication, and telomeres.

Chromosomes need origins of replication for DNA synthesis to occur

An origin of replication (ori) is a site at which DNA replication begins.  Origins of replication are often AT rich sequences, which allows them to be more easily pulled apart than GC rich sequences. Without an origin of replication, DNA cannot be replicated and will not be passed on to the next generation.

Eukaryotic chromosomes contain numerous replication origins, allowing for DNA synthesis to proceed in parallel at many sites simultaneously. Each pair of replication forks is referred to as a "replicon". Replicons can be visualized as bubbles of replicated DNA that expand in both directions. Eventually, all replicons merge into a single large bubble, until replication terminates at the telomeres.

The electron micrograph at right shows several replicons, each originating from a different origin (arrows).

Each replicon has two replication forks, moving in opposite directions. Ultimately, replication forks meet, until replication of each template strand is complete.

Displayed by hypertext link to http://bio3400.nicerweb.net/Locked/media/ch11/11_14-replication_bubbles.jpg

A series of experiments from Jack Szostak's lab helped to define the critical components of chromosomes. These experiments all had the same steps:

1. Transform yeast, deficient in Leucine production (Leu-)  with an artificial construct
2. Isolate a colony on complete media (permissive conditions)
3. Grow in complete media for several generations
4. Transfer to minimal media without Leucine
5. Identify clones that grow without Leucine

If you transfect into yeast a plasmid containing a selectable marker, in this case a LEU gene (Leucine biosynthesis) into Leu- yeast, the cells will not grow, even though you have given them the correct gene. But if you randomly clone yeast sequences into this same plasmid and select on minimal media, you can recover a few clones that are able to grow (ie. synthesize leucine). The inserts contained in these surviving plasmids are origins of replication, referred to in yeast as autonomously replicating sequences (ARS).
ARS have been identified in a variety of other species (eg. humans, Drosophila, maize, tobacco, even bacteria) by virtue of their ability to replicate in yeast.

Centromeres ensure equal division of genetic material between daughter cells


You might have noticed in the previous diagram that in the case of the ARS plasmid transformants, not all the daughter cells received the construct. Furthermore, in the absence of selection, even those cell lines may lose the construct. When yeast genomic DNA is randomly cloned into plasmids containing both a LEU gene and an ARS, some are stably inherited in the absence of selection. These plasmids happened to receive a fragment of DNA containing a centromere.
Centromeres are sequences of DNA to which the kinetochore proteins attach, and therefore to which spindle fibres attach during mitosis. Without a centromere, the construct has no guarantee of being passed on to both daughter cells during cell division. They tend to include repetitive sequences (satellite DNA), and also to not have many nucleosomes. Presumably, that is to make spindle fibre attachment during mitosis easier.

Detail of a positive staining centromere region of a C-banded human chromosome. (C banding preferentially stains constitutive heterochromatin). The fibrous organization of this region is still apparent but is either covered by, or embedded in, an amorphous matrix. Magnification x 64,000. Chromosomes and Chromatin, Vol. II (1988) Ed. K.W.Adolph. CRC Press. Fig. 14 pg. 67

Telomeres are needed for completion of replication, and protection, of the ends of chromosomes


In the first two experiments, the researchers created what are now known as Yeast Artificial Chromosomes (YACs). Up to now, we've been getting away with creating artificial chromosomes by making them circular. In yeast, they function as completely independent chromosomes. But eukaryotic chromosomes are linear, and Experiment C below illustrates that linear chromosomes can't normally replicate without telomeres at each end.
When Tetrahymena telomeric sequences were added to the ends of linearized yeast artificial chromosomes, the YACs were able to replicate and segregate stably in yeast.

Why is that? What function do telomeres serve that make them so necessary for linear chromosomes?

The basic problem with linear chromosomes are the ends - specifically, the 5' ends of a new strand. DNA polymerases can synthesize from 5' to 3' only. While the leading strand can read right to the end of the template, the lagging strand cannot. What ends up happening is this: Parent strands are in dark blue.

As you can see, the 5' ends of the daughter strands are short. Now, for one replication, this might not matter so much. The daughter cells would lose some DNA off the chromosome ends, but not too much. Probably, function would not be affected. But what happens in the next division, and the next? More and more DNA is lost and the chance of losing part of an important gene is much more likely. Put this way, a species with linear chromosomes and without telomeres is unlikely to survive for more than a few generations.

Prokaryotes avoid the problem of replicating the ends of linear molecules by instead having circular chromosomes

Circular chromosomes  satisfy the requirement that there must be an upstream DNA polymerase complex to remove RNA primers and fill in gaps.


Telomeres are specialized sequences that facilitate the replication of the ends of linear chromosomes and protect them from nuclease digestion.

Linear molecules will always have an unfilled gap upstream from the origin of replication closest to each end of the chromosome. Since this unfilled gap will always be a 3' protruding end, there is no way for DNA polymerase to "fill it in". Given that eukaryotes evolved so long ago, there must be a mechanism for dealing with this problem.

That solution is the telomere. Telomeric sequences are composed of repeated motifs and extend the linear eukaryotic chromosome. Think of telomeric sequences like a buffer for loss of sequence. As long as you add roughly as many nucleotides to the ends of chromosomes as they lose during each round of DNA replication, there is no net loss of DNA at the ends.

Telomeric sequence data was first obtained in Tetrahymena thermophilus, a cilliated protozoan. Tetrahymena has two separate nuclei, a germinal micronucleus, which carries a diploid complement of chromosomes, and a somatic macronucleus, in which particular chromosomes or parts of chromosomes are present in very high copy numbers. The rDNA genes are present on separate chromosomes with about 104 copies. Because the rDNA genes are located near the ends of the chromosomes, it was therefore possible to directly sequence the termini of chromosomal DNA using rDNA-specific primers. These studies revealed that individual rDNA termini carry a variable number (~120-420bps) of C4A2/T2G4 repeats.

Other examples:
  Oxytricha       C4A4
  Saccharomyces   C2-3A(CA)1-3
  Dictyostelium   C1-8T
H. sapiens CCCTAA

These telomeric sequences are oriented so that the C-rich strand always runs 5'-->3' from the end towards the interior of the chromosome. In some cases, there are nicks, or gaps in the C-rich strand.

Example: Oxytricha (ciliated protozoan):

             end of chromosome                 to centromere -->
5'            C4A4C4A4C4----------------------.....
3'  OH-G4T4G4T4G4T4G4T4G4----------------------.....

Telomeres actually have two functions:

Flourescent-labeled DNA fragments containing telomeric sequences were hybridized to metaphase chromosomes. Telomeric probe is visualized in white. Chromosomal DNA is counterstained with DAPI (blue).

Image displayed by hypertext link to The New Genetics, Chapter 2
RNA and DNA Revealed: New Roles, New Rules
Natl. Inst. of General Medical Science

Telomeres prevent degradation, repair and recombination of chromosome ends

The overhang ends are still a problem, though. Typically, the end of a linear DNA molecule would be recognized as a damaged piece of DNA and "repaired", resulting in loss of sequence from the ends of chromosomes, or fusion with other double-stranded DNAs. Eukaryotic cells have mechanisms for protecting chromosome ends from repair enzymes, which vary among eukaryotes. For example, telomeres of ciliates and fungi are protected by telomere binding proteins which effectively hide the telomeres from repair machinery.

Mammals have a more elaborate D-loop structure, in which the double-stranded telomere DNA opens up to form a single-stranded D-loop (or T-Loop). The 3' protruding end can loop back to form a T-loop. The end of the T-loop can base pair with internal repeat units by non-Watson-Crick base pairing. The D-loop is maintained through additional proteins that bind both the T-loop and the D-loop seen in the figure.

Telomerases carry a small RNA molecule which acts as a template for elongation of telomeric repeats.

Functionally, the telomere is a 3' protruding end which can not be elongated by DNA polymerase. Telomere terminal transferase (telomerase) allows the telomere to function as a 3' recessed end in the following way: The telomerase enzyme carries an RNA molecule (blue) whose sequence is the complement of the telomeric repeat. When the telomere (red) base pairs with this RNA, the 5' end of the RNA extends beyond the end of the telomere. This allows the RNA to act as template for the addition of nucleotides (green)to the telomeric 3' end, which acts as a primer. In the example, an RNA template with the sequence 5'(ccccaaaa)n3' codes for the actual telomeric repeat 5'(ttttgggg)n3'.

So, by extending the 3' ends of linear chromosomes with each round of DNA synthesis, telomerase provides a longer template for the lagging strand, which offsets the inevitable loss of DNA from the ends.

What do you think came first - telomeres or linear chromosomes? Why?

Telomerase activity varies with cell type and developmental stage

Is telomerase active in somatic cells? As it turns out, the answer depends on species is being observed, and stage of development. Below is an incomplete list of organisms along with telomerase activity in their somatic cells. Clearly there is much variation between eukaryotes when it comes to somatic telomerase activity.

Is telomerase active in somatic cells?

Unicellular eukaryotes - Telomerase is required in each cell division to maintain telomere length

1 - Telomerase activity is usually only seen in stem cells or germline cells, and telomerase activity is usually not found in somatic cells. It is hypothesized that because of the long human lifespan, somatic suppression of telomerase activity occurs as a check on cell proliferation, which could otherwise result in cancer. This is not a perfect control, because ultimately as telomeres are lost, oncogenes near the telomeres begin to be lost as well, resulting in cancer.

1 - Telomerase activity is often found in somatic cells in mice. This observation makes sense in contrast to the lack of telomerase activity in human somatic cells, because mice have very short life spans, and therefore would have less need for suppressing telomerase activity as a way of suppressing cancer.

Drosophila3 - doesn't use traditional short telomeric repeats elongated by telomerase. Instead, two retrotransposons, HeT-A and TART transpose specifically to chromosome ends, elongating the array of transposon repeats at the telomeres. (Weird or what?)

2 - High levels of telomerase activity was seen in actively dividing cells (roots and flowers), with low levels of activity in stems, and no detectable activity in mature leaves. This is consistent with the hypothesis that telomerase activity is needed in rapidly dividing tissues.

1 Wong JMY and Collins K (2003) Telomere maintanence and disease.  The Lancet 362:983-988.

2 Yang SW, Jin ES, Chung IK, Kim WT (2001) Expression of telomerase activity is closely correlated with the capacity for cell division in tobacco plants. J. Plant Biol. 44:168.

3 Danilevskaya ON, Traverse KL, Hogan NC, DeBaryshe GP and Pardue ML (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol. Cell. Biol. 19:873-881.

Some prokaryotes have linear chromosomes with telomere-like structures

Hinnebusch J, Tilly K (1993) Linear plasmids and chromosomes in bacteria. Mol. Microbiol. 10:917-922

Volff J-N, Altenbuchner J (2000) A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution. FEMS Microbiology Letters 186:143-150.

Ravin NV, Kuprianov VV, Gilcrease EB, Casjens SR (2003) Bidirectional replication from an internal ori site of the linear N15 plasmid prophage. Nucl. Acids Res. 31. DOI: 10.1093/nar/gkg856.

Surprising, right? Why are we talking about prokaryotes - don't they have circular chromosomes and therefore have no need for telomeres? In biology, every rule has exceptions. There are a number of cases in which prokaryotes have either linear chromosomes, or linear plasmids. One well-known example is bacteriophage Lambda, which is linear in its encapsidated form, but circularizes, by annealing of its "sticky ends", prior to replication as a circular molecule. However, there are now many examples of linear chromosomes and plasmids, including the spirochete Borrelia, the actinomycete Streptomyces, and the plant pathogen Agrobacterium.

see Figure 1 from Volff and Altenbucher, 2000.

While linear chromosomes or plasmids are rare amongst prokaryotes, their occurence in widely diverse species suggests that linearity has arisen independently numerous times over the course of microbial evolution. Two common schemes have been seen for linear chromosome replication: hairpins and invertrons.

Compare and contrast the methods used to deal with linear chromosomes by prokaryotes and by eukaryotes. Do you find that there is an underlying reason for the differences?