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Functional Genomics of Listeria Monocytogenes

Investigators
Minion, F. Chris
Institutions
Iowa State University
Start date
2001
End date
2003
Objective
The goal of this project is to construct microarrays of Listeria monocytogenes that represent serotypes 1/2a and 4b.
More information
NON-TECHNICAL SUMMARY: Listeria monocytogenes is a major food-borne pathogen that causes serious human illness. Completion of this project will enhance our fundamental understanding of the interactions of this pathogen under the environmental conditions encountered during its path from farm to food to human. These microarrays will allow scientists to study gene expression globally and better understand the role of specific genes under different environmental conditions.

APPROACH: The array consists of PCR products of 250-350 bp of each of the genes in the genome of Listeria monocytogenes. The 1/2a genome sequence was first made available by the Pasteur Institute and PCR primers were designed to amplify unique 250-350 base pair regions within each gene in this serotype. This was accomplished using Primer3 software and custom scripts to compare primers within the genome testing for uniqueness. The array also contains unique open reading frames (ORFs) of serotype 4b. These were identified by Blast comparison of the two genome sequences. The arrays will be validated by random DNA sequencing and by genomic hybridizations.

PROGRESS: 2002/01 TO 2002/12
The goal of this project is to construct microarrays of Listeria monocytogenes that represent serotypes 1/2a and 4b. The 1/2a genome sequence was first made available by the Pasteur Institute and PCR primers were designed to amplify unique 125-300 base pair regions within each gene in this serotype. This was accomplished using Primer3 software and custom scripts to compare primers within the genome testing for uniqueness. Over 2,800 PCR products have been amplified, purified and quality controlled, and are ready for spotting to the substrates. Ninety six of those represent unique genes in strain 4b. The remaining ORFs were either too small for inclusion in the array, coded for structural RNAs, or the PCR reactions failed for unknown reasons after several attempts with different primer pairs. We have also optimized spotting conditions for PRC products, and have screened several manufacturers for substrates with low background noise. Controls for the arrays have been constructed from mycoplasma gene sequences. These include both the PCR products for spotting as well as the RNA for inclusion in the labeling reactions.

IMPACT: 2002/01 TO 2002/12
Listeria monocytogenes is a significant food safety pathogen that is commonly found in the environment. Its growth at low temperatures is particularly troublesome because refrigeration does little to stop the growth of the organism. These microarrays will allow scientists to study gene expression on a global scale and come to a better understanding of the role of specific genes under different environmental conditions. This may lead to enhanced therapeutic strategies and reduced incidence of listeriosis.

PROGRESS: 2001/01/01 TO 2001/12/31
The goal of this project is to construct microarrays of Listeria monocytogenes that represent serotypes 1/2a and 4b. The 1/2a genome sequence was first made available by the Pasteur Institute and PCR primers were designed to amplify unique 25-300 base pair regions within each gene in this serotype. This was accomplished using Primer3 software and custom scripts to compare primers within the genome testing for uniqueness. Approximately 2,500 of the 2853 ORFs have been PCR amplified, purified and are ready for spotting to the substrates. The remaining 300 ORFs were either too small for inclusion in the array, coded for structural RNAs, or the PCR reactions failed for unknown reasons. We are redesigning primers for the failures in the group. The genome sequence for the 4b serotype is almost complete and a comparison of the two genomes revealed about 150 genes unique to the 4b serotype. We were able to design PCR primers for 96 of those ORFs and are presently amplifying those ORFs as well.

IMPACT: 2001/01/01 TO 2001/12/31
Listeria monocytogenes is a significant food safety pathogen that is commonly found in the environment. Its growth at low temperatures is particularly troublesome because refrigeration does little to stop the growth of the organism. These microarrays will allow scientists to study gene expression on a global scale and come to a better understanding of the role of specific genes under different environmental conditions. This may lead to enhanced therapeutic strategies and reduced incidence of listeriosis.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
IOWV-419-23-01
Accession number
193599
Categories
Parasites
Natural Toxins
Viruses and Prions
Bacterial Pathogens
Chemical Contaminants
Listeria