Abstract
Two human eukaryotic initiation factor 4E (eIF4E) genes were isolated and characterized from placental and chromosome 4-specific genomic libraries. One of the genes (EIF4E1) contained six introns, but the other gene (EIF4E2) was intronless, flanked by Alu sequences and 14-base pair (bp) direct repeats, and terminated by a short poly(A) stretch, all characteristics of retrotransposons. Numerous additional intronless eIF4E pseudogenes were found, but unlike EIF4E2, all contained premature in-frame stop codons. The entire EIF4E1 gene spanned >50 kilobase pairs. The coding regions of these two genes differed in four nucleotide residues, resulting in two amino acid differences in the predicted proteins. The promoter of EIF4E1 has been characterized previously. The putative promoter of EIF4E2 contained no TATA box but did contain a transcription initiator region (Inr) and numerous other sequence motifs characteristic of regulated promoters. EIF4E2 contained only two of the three polyadenylation signals present in EIF4E1. Evidence for transcription of both genes was obtained from primer extension, S1 mapping, ribonuclease protection, and reverse transcriptase-polymerase chain reaction experiments. Transcription was found to initiate 19 bp upstream of the translational initiation codon in the case of EIF4E1 and 80 bp in the case of EIF4E2. The two genes were differentially expressed in four human cell lines, Wish, Chang, K562, and HeLa.
Highlights
The best understood mechanisms for the regulation of protein synthesis involve modifications in the levels or activities of the initiation factors [1,2]
EIF4E levels are regulated at the transcriptional level. eIF4E mRNA is increased by overexpression of c-Myc as well as transformation of cells by v-Src and v-Abl [15]. eIF4E mRNA levels are elevated in a variety of cells that have been oncogenically transformed by in vivo transfection, viral infection, or chemical mutagenesis [16]
For both EIF4E1 and EIF4E2, the numbering system is based on human eIF4E cDNA [35], i.e. the location of the first ATG in the coding region is designated ϩ1, with upstream nucleotides having negative numbers and downstream nucleotides positive numbers
Summary
Materials—Two human placental genomic libraries in bacteriophage vectors (EMBL3-SP6/T7 and FIX II) were purchased from CLONTECH (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively. Three human chromosome 4-specific genomic libraries in Charon 21 (LLO4NSO2 and LAO4NSO1) and Charon 40 (LAO4NLO1) and one chromosome 20-specific genomic library in Charon 21 (LL20NSO1) were purchased from the ATCC (Rockville, MD). Bacterial strains NM538 and LE392 were purchased from Stratagene (La Jolla, CA). Restriction endonucleases and the DNA Cycle Sequencing System were purchased from Promega (Madison, WI). Radioisotopes (Ͼ5000 Ci/ mmol) were purchased from ICN (Costa Mesa, CA). The Geneclean kit was purchased from BIO 101, Inc.
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