Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

Journal Article · Thu Mar 01 04:00:00 EST 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.6.2206· OSTI ID:6073643

Monaco, L; Murtagh, J J; Newman, K B; Tsai, Su-Chen; Moss, J; Vaughan, M ^[1]

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1. National Institutes of Health, Bethesda, MD (USA)

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

• OSTI ID: 6073643
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 87:6; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACID SEQUENCE
ANTIGENS
AROMATICS
AZAARENES
BACTERIAL DISEASES
CHEMICAL ACTIVATION
CHOLERA
CYCLASES
DISEASES
DNA HYBRIDIZATION
DNA SEQUENCING
ENZYMES
GENE AMPLIFICATION
GENES
GUANINE
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
HYDROXY COMPOUNDS
INFECTIOUS DISEASES
LYASES
MATERIALS
MESSENGER-RNA
MOLECULAR STRUCTURE
NUCLEIC ACIDS
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PURINES
RNA
STRUCTURAL CHEMICAL ANALYSIS
TOXIC MATERIALS
TOXINS
TRANSCRIPTION