Gene structure and enzymatic activity of mouse eosinophil-associated ribonuclease 2.
Mouse eosinophil-associated ribonuclease-2 (mEAR-2) is one of a cluster of genes identified in the genome of the mouse Mus musculus that are highly divergent orthologs of the primate ribonucleases, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Northern analysis revealed expression of genes hybridizing to mEAR-2 in mouse lung, liver and spleen tissues. We obtained full-length cDNA by hybridization screening of mouse eosinophil and lung cDNA libraries and by rapid amplification of cDNA ends (RACE) from liver, spleen and lung RNA. Using these methods we have isolated the 195 base pair (bp) 3' untranslated region (UTR) that includes a typical polyadenylation signal preceding a poly A tail and the 5' UTR which includes 63-71 bp and three distinct transcriptional start sites. Using unidirectional PCR we isolated a 361-bp 5' promoter region and delineated the intronic / exonic boundaries which include a non-coding exon 1, a single intron, and a coding exon 2, a structure that is typical of genes of the RNase A superfamily. Consensus sites for PU.1 and EoTF, both active as intronic enhancer elements of the gene encoding EDN, are also present in the intron of the gene encoding mEAR-2. The catalytic activity of recombinant baculovirus-derived mEAR-2 is similar to that of rhEDN from this source, with catalytic constants k(cat)/K(m)=5.6x10(6) M(-1) s(-1) and 10.5x10(6) M(-1) s(-1), respectively, against a standard yeast tRNA substrate. Sequence analysis of the non-coding regions and enzymatic characterization of the gene product provide further evidence indicating that mEAR-2 is a structural and functional ortholog of primate EDNs and ECPs.
McDevitt
A L
AL
Eosinophil Biology Unit, Laboratory of Host Defenses 10/11N104, NIAID / National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1886, USA.
Deming
M S
MS
Rosenberg
H F
HF
Dyer
K D
KD
D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D047091:Eosinophil Granule Proteins; D047208:Eosinophil-Derived Neurotoxin; D005796:Genes / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014158:Transcription, Genetic
D001798:Blood Proteins; D018076:DNA, Complementary; D047091:Eosinophil Granule Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases