CENTROMERES

The centromere is specifically concerned with chromosomal disjunction and segregation. There are sequences within the DNA and associated protein components giving a structure ~400 nm in diameter, known as the KINETOCHORE.

Centromere is a region of a chromosome responsible for segregation at mitosis and meiosis, serve as attachment site for spindle fibres and is site at which spindle fibres shorten causing chromosomes to move to the poles.

The DNA sequences were first of interest in establishing whether DNA molecules possessing these sequences can segregate properly at cell division or if absent would lead to instability.

Hence yeast replicative plasmids (YRPs) have been constructed that possess a yeast origin of replication. These plasmids can replicate autonomously but are extremely unstable at mitosis and meiosis and disappear from cells, unless selection is applied, because they segregate erratically.

DNA sequences from yeast libraries when cloned into such plasmids have enabled detection of sequences that can confer mitotic stability on YRPs. And minimum size sequences necessary for centromere function have been established and analysed.

These share common features and can be swapped between chromosomes in yeast with unimpaired function, i.e. they play no role in distinguishing one chromosome from another. However, centromere sequences are highly species-specific.

Centromeres discovered 1980.

In the yeast centromere (CEN) sequences there are three major domains called CENTROMERE DNA SEQUENCE ELEMENTS (CDEs) I, II & III.

CDE I

- 9 bp sequence: 5'-T G A C A T G A T-3' or very similar

- highly conserved, minor variations at left boundary.

CDE II

- a block of 70-90 bp of DNA sequence that is >90% A+T rich.

CDE III

- a 11 bp sequence, highly conserved

e.g. T G A T T T C C G A A - mutations in the CCG triplet totally inactivate centromere function.

E.g. CEN 3:

TCACATGAT*GATATTTGATTTTATTATA//ATTAGTGTATT*TGATTTCCGAA

TELOMERES

At ends of chromosomes, conferring stability on linear chromosomes.

Also function to:

distinguish chromosome ends from breaks as broken ends tend to be ‘sticky’

mediate chromosome to chromosome interaction

mediate interaction of chromosome ends with nuclear envelope (capable of binding to nuclear membrane)

protect against exonucleases

allow replication of the chromosome end of the lagging strand.

Telomere sequences are similar in eukaryotes (highly conserved). They are long arrays of the general form

5' C_n (A / T)_m 3' where n > 1 and 1 # m # 4

e.g. Human telomere sequence is (TTAGGG)_n

or 5'C₃TA₂ - several kb of this sequence at end of chromosome.

Telomeric sequences in selected organisms:

How are telomeric repeats synthesized?

Extracts of Tetrahymena contain an enzyme TELOMERASE that uses the 3'OH end of the G+T telomeric sequence as a primer for synthesis of tandem T₂G₄. Only dTTP and dGTP are required.

Telomerase is a ribonucleoprotein. It contains a short RNA component 159 bases long in Tetrahymena. Each RNA molecule includes a 15-22 base sequence identical to two repeats of the C+A/T telomeric sequence (with U instead of T) which is used as a template for extending the G+T strand.

Bases are added individually in the correct sequence. The process is discontinuous:

RNA template (in telomerase) is positioned on the DNA primer, several nucleotides complementary to template are added, enzyme translocates to the new 3' end and the process is repeated.

There would seem to be a 3' overhang which may loop back to form a hairpin end to the molecule with G-G complexes forming.

Telomeres seem to be undergoing continuing lengthening and shortening. Normal human somatic cells express very low or no detectable telomerase activity and progressively lose their telomeric sequences with age. In contrast, germline cells and most tumor cells have high telomerase activity.

Tetrahymena telomeres function in yeast and after several replications, yeast telomeres are added on.

The RNA component of human telomerase has been cloned and sequenced, and the template region is

5' C U A A C C C U A A C 3'

complementary to human telomere sequence (5' TTAGGG 3')_n

(i) Examine the need for a special replication need at ends of (linear) chromosomes.

(ii) What genetic elements are necessary for a stable artificial chromosome? Hint: examine yeast artificial chromosomes (YACs) used as cloning vectors.
 

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Blackburn & Szostak (1984) Annual Review of Biochemistry 53, 163-94.

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