Which of the following is absent in YAC Satellite,ORI, centromere, telomere
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A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d’Étude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human β-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.
As the time rapidly approaches when the complete sequence of a human chromosome will be known, it is striking how little is known about how human chromosomes function. In contrast, the necessary elements for chromosomal function in yeast have been defined for several years. Three important elements appear to be required for the mitotic stability of linear chromosomes: centromeres, telomeres, and origins of replication. The ascertainment of these elements in Saccharomyces cerevisiae provided the basis for the construction of yeast artificial chromosomes (YACs), which have proven to be important tools both for the study of yeast chromosomal function and as large capacity cloning vectors (1–3).
The use of a similar strategy in human cells to produce human artificial chromosomes (HACs) might be expected to provide an important tool for the manipulation of large DNA sequences in human cells. However, of the three required chromosomal elements, only telomeres have been well defined in human cells to date. It has been demonstrated that telomeric DNA, consisting of tandem repeats of the sequence T2AG3, can seed the formation of new telomeres when reintroduced into human cells (4–6). And recently, two telomeric binding proteins, TRF1 and TRF2, have been described (7–9). The second required element, a human centromere, is thought to consist mainly of repeated DNA, specifically the alpha satellite DNA family, which is found at all normal human centromeres (10–12). However, normal human centromeres are large in size and complex in organization, and sequences lacking alpha satellite repeats also have been shown to be capable of human centromere function (13, 14). As for the third required element, the study of origins of DNA replication also has led to conflicting reports, with no apparent consensus sequence having yet been determined for the initiation of DNA synthesis in human cells (15, 16).
The production of HACs from cloned DNA sources should help to define the elements necessary for human chromosomal function and to provide an important vector suitable for the manipulation of large DNA sequences in human cells. Two approaches to generate chromosomes with the “bottom up” strategy in human cells from human elements have recently been described. Harrington et al. (17) synthesized arrays of alpha satellite DNA, which were combined in vitro with telomeres and fragmented genomic DNA, and transfected into HT1080 cells. The undefined genomic DNA component appeared to play an important role in the ability to form HACs, leaving unanswered questions as to what sequences, other than telomeres and alpha satellite DNA, were necessary for chromosome formation. Ikeno et al. (18) used two 100-kb YACs containing alpha satellite DNA from human chromosome 21 propagated in a recombination deficient strain, which necessitated transient expression of a recombination protein (Rad52) to modify the YAC with telomere sequences and selectable markers (19). Only one of the two YACs was able to form HACs in HT1080 cells, suggesting that not all alpha satellite sequences may be able to form centromeres.
Here we report construction of functional HACs from a YAC that was propagated in a recombination-proficient yeast strain and was chosen solely for its size (1 Mb) and the presence of alpha satellite DNA. This YAC contains both alpha satellite and non-alpha satellite DNA and was modified to include a putative human origin of replication and human telomeric DNA. The function and stability of HACs generated from this 1-Mb YAC in a human cell line are described.
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