PMC

Alignment of the putative novel DNA helicase to Rad3/ERCC2 subfamily of DNA helicases. The multiple sequence alignment was performed by using the program clustalw Ver. 1.74 and enhanced to display identical amino acid residues in shaded green and similar residues in shaded blue. In addition, the seven conserved domains of known DNA helicases are shown above the alignment, and the nucleotide- and DNA-binding domains are shown below the alignment.

Genomic structure of M68 and the adjacent genes. (A) Exon/intron organization of human M68 BAC genomic sequence. Exons, which are represented as shaded boxes, were determined by using the crossmatch program and manual editing to conform exon boundaries to standard splice-site consensus sequences. Arrows represent the translation initiation methionine and the direction of protein translation. (B) Schematic representation of the M68 gene cluster and family structure.

IHC scoring of M68 overexpression in formalin-fixed paraffin-embedded sections from GI-tract cancer tissues. (A) No or minimal staining, in <10% of the tumor cells; score, 0. (B) Faint barely visible staining in >10% of the tumor cells; score, 1+. (C) Weak to moderate staining in >10% of the tumor cells; score, 2+. (D) Strong staining in >10% of the tumor cells; score, 3+. 2+ and 3+ are considered positive for M68 overexpression.

Expression pattern of M68 mRNA in human tissues and tumors. (A) For each tissue type, mRNA was isolated from tumors from three different donors and from a normal control tissue. T, tumor tissues; N, normal tissues. The same blot was hybridized to a human ubiquitin probe to verify RNA loading in each lane. (B) Human cancer cell lines (PBL, peripheral blood leukocytes). (C) Normal human tissues.

M68 gene amplification and overexpression. (A and B) A representative of a M68-overexpressing gastrointestinal tumor without M68 gene amplification. (A) FISH dot count with M68 BAC DNA. (B) IHC of M68 protein expression to the adjacent section of A. (C and D) A representative of M68-overexpressing gastrointestinal tumor with M68 gene amplification. (C) FISH dot count with M68 BAC DNA. (D) IHC of M68 protein expression to the adjacent section of C. (E) TaqMan quantitative PCR analysis of M68 genomic DNA. Samples c and f were from the same individual tumors as shown in A, B and C, D, respectively. Data shown are the average of at least two independent experiments.

IHC of M68 and FAS in normal and tumor tissues of GI tract. Note strong positive staining in malignant epithelial cells (arrows) in tumor samples (A, colon; F, esophagus; G, stomach; and H, rectum). In normal adjacent colon (E), weak M68 expression was detected in epithelial cells (arrows) lining the lumen and was generally absent in the glandular epithelium. A significant decrease in staining was observed when M68 antibody was preincubated with the immunizing peptide (B), and no tumor epithelial cell staining was observed in tumor tissues with preimmune serum (C) or a nonimmune rabbit serum (D). IHC of Fas (CD95) (I) and M68 (J) in a colon adenocarcinoma. Note the similar staining pattern and coexpression of CD95 and M68 in the tumor epithelial cells (arrows).