ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.
BACKGROUND
Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples.


METHODS
Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples.


RESULTS
After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1.


CONCLUSIONS
In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.
Tian
Wenzhi
W
Department of Medicine, New York University School of Medicine, North Shore University Hospital,and the North Shore-LIJ Research Institute, Manhasset, NY 11030, USA.
Chua
Kevin
K
Strober
Warren
W
Chu
Charles C
CC
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics; D001435:Bacteriophages / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D015415:Biomarkers / Q000737:chemistry*; D002478:Cells, Cultured; D016384:Consensus Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005798:Genes, Bacterial; D006801:Humans; D019426:Integrases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D045522:Recombinases; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D015415:Biomarkers; D004269:DNA, Bacterial; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; C472972:ILG1 protein, E coli; D019426:Integrases