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

Abstract

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 frommore » 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.« less

Authors:
; ; ; ; ;  [1]
  1. (National Institutes of Health, Bethesda, MD (USA))
Publication Date:
OSTI Identifier:
6073643
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA); Journal Volume: 87:6
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Monaco, L., Murtagh, J.J., Newman, K.B., Tsai, Su-Chen, Moss, J., and Vaughan, M. Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin. United States: N. p., 1990. Web. doi:10.1073/pnas.87.6.2206.
Monaco, L., Murtagh, J.J., Newman, K.B., Tsai, Su-Chen, Moss, J., & Vaughan, M. Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin. United States. doi:10.1073/pnas.87.6.2206.
Monaco, L., Murtagh, J.J., Newman, K.B., Tsai, Su-Chen, Moss, J., and Vaughan, M. Thu . "Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin". United States. doi:10.1073/pnas.87.6.2206.
@article{osti_6073643,
title = {Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin},
author = {Monaco, L. and Murtagh, J.J. and Newman, K.B. and Tsai, Su-Chen and Moss, J. and Vaughan, M.},
abstractNote = {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.},
doi = {10.1073/pnas.87.6.2206},
journal = {Proceedings of the National Academy of Sciences of the United States of America; (USA)},
number = ,
volume = 87:6,
place = {United States},
year = {Thu Mar 01 00:00:00 EST 1990},
month = {Thu Mar 01 00:00:00 EST 1990}
}