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 00:00:00 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), Vol. 87:6; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

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