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Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Identification of four novel developmentally regulated gamma hemoglobin mRNA isoforms.
Experimental Hematology 2009 Februrary
OBJECTIVE: In the course of developing assays for the molecular prediction of biological age, we serendipitously discovered four novel isoforms of gamma hemoglobin mRNA, designated HBG1n1, HBG1n2, HBG2n2, and HBG2n3, collectively termed HBGn isoforms. Here we report the molecular characterization and tissue expression of these isoforms.
MATERIALS AND METHODS: RNA obtained from human peripheral blood and various fetal and adult tissues was amplified with duplex reverse transcription polymerase chain reaction (RT-PCR) assays to determine the expression profiles of the HBGn isoforms. To determine if their molecular origin was either a genomic recombination or an undefined RNA rearrangement event, DNA and RNA samples were pretreated with RNaseI and/or DNaseI and then analyzed with the duplex RT-PCR assays.
RESULTS: Alignment analysis indicated that the HBG1n(1/2) and HBG2n(2/3) isoforms were identical to the expected parental HBG1 or HBG2 sequences except for unique deletions that spanned the 3' end of exon two, the entire second intron, and the 5' end of exon three. RT-PCR duplex reactions revealed that the isoforms exhibit restricted expression to fetal hematopoietic tissue and newborn peripheral blood and appear to be temporally regulated during development. Finally, nucleic acid digestion experiments illustrate that the isoforms appear to be created by an RNA rearrangement event between penta- or octanucleotide direct repeats in adjacent exons.
CONCLUSION: The precise genesis and function of these novel isoforms is unknown at present. Interestingly, each of the four isoforms identified maintain an open reading frame as well as 5' and 3' regulatory regions invoking the possibility that they encode a family of related polypeptides.
MATERIALS AND METHODS: RNA obtained from human peripheral blood and various fetal and adult tissues was amplified with duplex reverse transcription polymerase chain reaction (RT-PCR) assays to determine the expression profiles of the HBGn isoforms. To determine if their molecular origin was either a genomic recombination or an undefined RNA rearrangement event, DNA and RNA samples were pretreated with RNaseI and/or DNaseI and then analyzed with the duplex RT-PCR assays.
RESULTS: Alignment analysis indicated that the HBG1n(1/2) and HBG2n(2/3) isoforms were identical to the expected parental HBG1 or HBG2 sequences except for unique deletions that spanned the 3' end of exon two, the entire second intron, and the 5' end of exon three. RT-PCR duplex reactions revealed that the isoforms exhibit restricted expression to fetal hematopoietic tissue and newborn peripheral blood and appear to be temporally regulated during development. Finally, nucleic acid digestion experiments illustrate that the isoforms appear to be created by an RNA rearrangement event between penta- or octanucleotide direct repeats in adjacent exons.
CONCLUSION: The precise genesis and function of these novel isoforms is unknown at present. Interestingly, each of the four isoforms identified maintain an open reading frame as well as 5' and 3' regulatory regions invoking the possibility that they encode a family of related polypeptides.
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