title: Segments missing from the draft human genome sequence can be isolated by transformation-associated recombination cloning in yeast.
abstract: The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were re-isolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E. coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.
authors.affiliation: Laboratory of Biosystems and Cancer, National Cancer Institute, Building 37, Room 5031, Bethesda, Maryland 20892, USA. kouprinn@mail.nih.gov
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Leem
authors.forename: Greg
authors.initials: G
authors.lastname: Solomon
authors.forename: Albert
authors.initials: A
authors.lastname: Ly
authors.forename: Maxim
authors.initials: M
authors.lastname: Koriabine
authors.forename: John
authors.initials: J
authors.lastname: Otstot
authors.forename: Eugene
authors.initials: E
authors.lastname: Pak
authors.forename: Amalia
authors.initials: A
authors.lastname: Dutra
authors.forename: Shaying
authors.initials: S
authors.lastname: Zhao
authors.forename: J Carl
authors.initials: JC
authors.lastname: Barrett
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
mesh_terms: D015815:Cell Adhesion Molecules / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins; D015894:Genome, Human; D016045:Human Genome Project; D006801:Humans; D017436:Kallmann Syndrome / Q000235:genetics; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: C076266:ANOS1 protein, human; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D004247:DNA