Several studies have shown a high degree of

Several studies have shown a high degree of sequence conservation especially in the nucleic Polymyxin B sulfate binding domain, usually referred to as `cold shock domain or CSD,\' highlighting the importance of the metabolic processes regulated by Y-box proteins , , , , . A direct role for YB-1 in cell division and cell proliferation has been suggested, based on the following observations. (i) YB-1 is known to activate several cell proliferation-related genes such as proliferating cell nuclear antigen (PCNA), DNA polymerase, thymidine kinase , epidermal growth factor receptor, and growth hormone receptor genes , (ii) YB-1 is expressed at higher levels in actively proliferating cells compared to quiescent cells , , and (iii) YB-1 is over-expressed in regenerating liver and in liver cancers whereas in normal adult liver it is barely detectable , . However, direct evidence is lacking. DT40 cells have been reported to undergo homologous recombination at an exceptionally high frequency , . This property has earlier been exploited for targeted disruption of a number of genes , , , . To gain a better insight into the function of YB-1, we disrupted one allele of the Chk-YB-1b gene and analyzed the resulting phenotype of the heterozygous DT40 cells (DT40-YB-1b). Compared to wild-type DT40 cells, DT40-YB-1b cells have an increased cell size, increased genomic DNA content (4), and grow slowly (doubling time of approximately 35–40h compared to 12–13h for wild-type cells), resembling phenotypic abnormalities typical of cells with a defect in G2/M. We have shown further that a fraction of these mutant cells undergo apoptosis. Thus, our results provide, for the first time, direct evidence for the crucial role of YB-1 in cell growth. Materials and methods Construction of the Chk-YB-1b-Neo vector. Approximately 107 plaques from a chicken genomic library established in EMBL3 vector (Clontech Palo Alto, CA) was screened and one clone containing a 12kb insert was selected. Digestion of this clone with SalI and EcoRI restriction enzymes released five different fragments of 4.5, 2.5, 2.1, 1.8, and 0.6kb. Southern hybridizations indicated that the 2.1, 1.8, and 0.6kb fragments carried sequences corresponding to the Chk-YB-1b cDNA. Therefore, these fragments were cloned into the plasmid pGEM-3Z vector and sequenced. The pCINeo plasmid DNA (Promega, Madison, WI) was digested with SspI and BamHI, and the 1.4kb fragment containing the SV40-NeoR (neomycin-resistance) gene was eluted and end-filled with Klenow. pGEM-Chk-YB-1b was digested with StyI, the large fragment was eluted, end-filled with Klenow, and ligated with the 1.4kb SV40-NeoR fragment using T4 DNA ligase. Following transformation of Escherichia coli Top10 F+ competent cells, appropriate clones were selected and DNAs were isolated. Transfection and selection of G418-resistant DT40 cells. DT40 cells [18], [19], obtained from Dr. Koi, National Institute of Environmental Sciences, Durham, NC, were grown at 37°C in a humidified incubator containing 5% carbon dioxide in DMEM (high glucose) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), 10% tryptose phosphate broth, 2% chicken serum (Sigma, St. Louis, MO), penicillin, and streptomycin. Cells were sub-cultured every two days. About 5×105 DT40 cells in mid-log phase of growth were transfected with 5.0μg pGEM3Z-Chk-YB-1b-Neo DNA using 25μl LipofectAMINE (Life Technologies, Rockville, MD) following the protocol suggested by the manufacturer. Eighteen hours after transfection, cells were transferred to the selection medium containing 700μg/ml G418. Selection medium was changed every 48h. After 25 days of selection, the G418-resistant DT40 cells were sub-cloned by diluting cultures to about 1cell/10ml and plating 1ml per each well of two 96-well plates. After 10 days, we found that G418-resistant cells began to grow in 20 different wells, which were numbered as Chk-YB-KO1–20.