TY - JOUR
T1 - Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability
AU - Sleister, Heidi Major
AU - Mills, Kathleen A.
AU - Blackwell, Sue E.
AU - Killary, Ann M.
AU - Murray, Jeffrey C.
AU - Malone, Robert E.
N1 - Funding Information:
We thank B.Weiffenbach for the YAC B22F9. Also, we are grateful to Anne Galbraith and Doug Pittman for reviewing the manuscript. Amy Greene assisted in screening the YAC library to find human clones and also reviewed the manuscript. This work was supported by an NIH ROI grant (#HGOO355) to J.C.M. and R.E.M.
PY - 1992/7/11
Y1 - 1992/7/11
N2 - In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.
AB - In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.
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U2 - 10.1093/nar/20.13.3419
DO - 10.1093/nar/20.13.3419
M3 - Article
C2 - 1630913
AN - SCOPUS:0026690839
SN - 0305-1048
VL - 20
SP - 3419
EP - 3425
JO - Nucleic acids research
JF - Nucleic acids research
IS - 13
ER -