mRNA differential display of colonic mucosa cells in ulcerative colitis.
Involvement of mucosal cells in inflammatory bowel disease (IBD) may be a sequenced process and the molecular difference between involved and uninvolved cells might implicate a possible mechanism in the disease process. The aim of this study was to compare gene expression between involved and uninvolved colonic mucosa cells in an individual with ulcerative colitis (UC) and to clone, sequence, and identify those differentially expressed genes, mRNA differential display was used to identify the gene expression in the mucosa of the UC patient. Anchored oligo(dT) primers and random 5' oligonucleotide 10-mer were used to carry out polymerase chain reaction on reverse-transcribed RNA (RT-PCR). The amplified cDNAs were displayed on a standard sequencing gel and comparisons were drawn between each two lanes representing either involved or uninvolved cells from a specific combination of two primers. Wherever differences were noted between lanes, the bands were reamplified using PCR, cloned into specialized vectors for positive selection, and then confirmed by dot blot. The cloned genes were then sequenced and compared with the GenBank database. About 1200 mRNA species were displayed in the sequencing gel. Among them, 106 fragments were differentially expressed between the two groups. Twenty-five of those differentially displayed gene fragments have been isolated, reamplified, and cloned for sequencing and dot blot analysis. Seventeen of the fragments were differentially expressed using the dot blot technique. Among those 25 gene fragments, 14 have homology to known genes and 11 have no match to any reported genes. Those matched known genes included genes for parathyroid tumor, T cell receptor-beta, alpha-nascent polypeptide-associated complex, ovarian cancer, and myeloblast. This is the first study using mRNA differential display to observe differential gene expression between involved and uninvolved mucosa cells in UC and it shows that differential display is a rapid method for characterizing gene changes in vivo in ulcerative colitis. The results from this study may provide useful information and facilitate further gene studies in this disease.
Zuo
L
L
Department of Surgery, University of Cincinnati, Ohio 45267, USA.
Ogle
C K
CK
Fischer
J E
JE
Nussbaum
M S
MS
D001345:Autoradiography; D001483:Base Sequence; D003001:Cloning, Molecular; D003093:Colitis, Ulcerative / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology*; D003106:Colon / Q000473:pathology / Q000503:physiopathology*; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology / Q000503:physiopathology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger