Distinction of mouse interferon-alpha subtypes by polymerase chain reaction utilizing consensus primers and type-specific oligonucleotide probes. 1994

T K Hughes, and R Chin, and S K Tyring, and P L Rady
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555-1019.

We have developed a novel method to study the subtype-specific expression of interferon-alpha (IFN-alpha) in the mouse system: we synthesized and used consensus oligonucleotide primers to allow simultaneous polymerase chain reaction (PCR) amplification of multiple murine IFN-alpha gene sequences. In addition, a set of subtype-specific oligomer probes were designed and used to distinguish between IFN-alpha genes that differ by only a few bases. The consensus primers, corresponding to two regions highly conserved among murine IFN-alpha subtypes, were used in reverse transcription and PCR amplification of total cellular RNA, isolated from IFN-gamma-treated murine L-929 cells, to yield a fragment of the anticipated approximately 520-bp size. Southern analysis of the amplified product using an internal consensus oligomer probe confirmed specific amplification of murine IFN-alpha gene(s). Subtype-specific oligonucleotide probes indicate that IFNs-alpha 1, -alpha 2, and -alpha 5 are present following IFN-gamma treatment, whereas IFN-alpha 4 remains virtually absent. Our results indicate that the expression of specific IFN-alpha subtypes may be subject to complex regulation, dependent upon inducing agents and cell types involved, and countless other factors. The procedure described here represents a novel method for studying the subtleties of the murine IFN-alpha mRNA expression.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012313 RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed) RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D015345 Oligonucleotide Probes Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin. Oligodeoxyribonucleotide Probes,Oligonucleotide Probe,Oligoribonucleotide Probes,Probe, Oligonucleotide,Probes, Oligodeoxyribonucleotide,Probes, Oligonucleotide,Probes, Oligoribonucleotide
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016384 Consensus Sequence A theoretical representative nucleotide or amino acid sequence in which each nucleotide or amino acid is the one which occurs most frequently at that site in the different sequences which occur in nature. The phrase also refers to an actual sequence which approximates the theoretical consensus. A known CONSERVED SEQUENCE set is represented by a consensus sequence. Commonly observed supersecondary protein structures (AMINO ACID MOTIFS) are often formed by conserved sequences. Consensus Sequences,Sequence, Consensus,Sequences, Consensus
D016898 Interferon-alpha One of the type I interferons produced by peripheral blood leukocytes or lymphoblastoid cells. In addition to antiviral activity, it activates NATURAL KILLER CELLS and B-LYMPHOCYTES, and down-regulates VASCULAR ENDOTHELIAL GROWTH FACTOR expression through PI-3 KINASE and MAPK KINASES signaling pathways. Interferon Alfa,Interferon, Leukocyte,Interferon, Lymphoblast,alpha-Interferon,IFN-alpha D,IFN-alpha5,Interferon alpha-1,Interferon alpha-17,Interferon alpha-4,Interferon alpha-5,Interferon alpha-7,Interferon alpha-88,Interferon alpha-J,Interferon alpha-T,Interferon alpha4,Interferon alpha5,Interferon, Lymphoblastoid,Interferon, alpha,LeIF I,LeIF J,Leif D,IFN alpha D,IFN alpha5,Interferon alpha,Interferon alpha 1,Interferon alpha 17,Interferon alpha 4,Interferon alpha 5,Interferon alpha 7,Interferon alpha 88,Interferon alpha J,Interferon alpha T,Leukocyte Interferon,Lymphoblast Interferon,Lymphoblastoid Interferon,alpha Interferon
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus

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