Molecular cloning and characterization of a rabbit eIF2C protein.
Rabbit eIF2C (94kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. Based on the partial amino acid sequences of Endolys C cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding cDNA fragment from a rabbit liver cDNA library. This fragment was subsequently used to screen for larger cDNAs. Marathon cDNA amplification and 5'-rapid amplification of cDNA ends were used to confirm the translation start site. Sequences from the overlapping clones were assembled into a 3599-bp composite sequence, which contains a single open reading frame that translates into a 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94kDa eIF2C detected a higher-molecular-weight polypeptide (140kDa). No 94kDa polypeptide was detected. The cloned cDNA was further characterized by in-vitro transcription-coupled translation in reticulocyte lysate. The translated product was precipitated with antibodies against eIF2C. Genomic Southern blot analysis indicates that the rabbit eIF2C is a single copy gene. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans.
Zou
C
C
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
Zhang
Z
Z
Wu
S
S
Osterman
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005796:Genes / Q000235:genetics; D016678:Genome; D020662:Guanine Nucleotide Exchange Factors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012333:RNA, Messenger; D004247:DNA