Abstract
A complete cDNA clone encoding Euglena gracilis chloroplast translational initiation factor 3 (IF-3chl) has been obtained. Analysis of the sequence indicates that the IF-3chl mRNA contains the spliced leader found at the 5' end of nuclear encoded mRNAs in E. gracilis. The open reading frame for IF-3chl encodes a 537-amino acid protein. IF-3chl appears to be divided into four domains. The first 140 amino acids correspond to a transit peptide required for the import of IF-3chl into the chloroplast. The mature form of IF-3chl encompasses domains 2-4 and is about twice the size of Escherichia coli IF-3. The second domain has no homology to other known proteins. It begins with a stretch of 35 residues, of which about 30% are proline. Downstream from this region is a stretch of about 25 amino acids with a repeating (GX)n motif followed by a very acidic region. The third domain comprises a region of about 175 residues and has between 31 and 37% homology to the IF-3s found in other organisms. The IF-3 homology domain is followed by an acidic region which has no detectable homology to other sequences. Analysis of E. gracilis genomic DNA suggests that there are about four copies of the IF-3chl gene, one of which is probably a pseudogene. The activity of IF-3chl is inducible by light. However, the IF-3chl mRNA is present in approximately equal amounts in both dark- and light-grown cells, suggesting that the light-dependent induction of IF-3chl activity is post-transcriptional.
Highlights
Qiong L i d, Lan Ma$, WilliamBurkharts, and Linda L
The third domain com- tures and functions of the corresponding prokaryotic factors. prises a region of abo1u7t5 residues and has between31 IF-3, is physically unusual compared to the bacteand 37% homologtyo the IF-3sfound in other organisms.rial factor.E. coli IF-3is a monomeric protein witha molecular The IF-3 homology domain is followed by an acidic re- mass of 22kDa
Techniques commonly used to lo6independent recombinants, while the oligo(dT)-library conta5inxed concentrate and change the buffer of a protein sample lead to lo4 independent plaques. The latter library was amplified in E. coli significant losses ofIF-3, even when siliconized tubes were
Summary
It binds to the 30S subunit and inhibits its association Materials-Oligonucleotide primers weremade in the Lineberger with the 50 S subunit. Bacillaris Cori (E.gracilis B) and the light induction of chloroplast development wercearried out basically as. Lecular mass 42 kDa) was purified as described [3], except that the DNA Sequencing-All the putative clones weresequenced by the TSKgel heparin-5PW column was omitted. RNA and DNA were transferred from the agarose gels to cleavagetime.Afterdigestion,peptideswereseparated by reverse Zeta-Probe blotting membranes as recommended by the manufacturer. Phase high performance liquid chromatography usinga Hypersil ODS For hybridization reactiontshe blotted membranes were incubated with column (2.1x 100 mm; Hewlett-Packard) employing a linear gradoifent 2 x lo cpm of 32P-labeledprobe (2 x lo cpdpg ) i n 1mM EDTA, 0.5 M. This washing procedure was repeated prior to reprobing the DNA was removed by lithium chloride precipitation.Poly(AY RNAwas blot.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
