Abstract
The yeast ribosome contains three acidic proteins, L44, L44', and L45, closely related from a structural point of view, that seem to play a functional role similar to that of proteins L7 and L12 in the bacterial ribosome. By screening a cDNA bank in lambda gt11 with specific polyclonal and monoclonal antibodies, recombinant phages expressing each one of the acidic proteins have been cloned. A unique copy of each gene is detected using the phage cDNA inserts as probes in nitrocellulose blots of yeast DNA digested with different restriction enzymes. The inserts were subcloned in the plasmid pUC19, and their physical maps and nucleotide sequences were determined. By using the cDNA inserts as probes in genomic DNA banks, DNA fragments carrying the acidic protein genes have been cloned, characterized, and sequenced. The results conclusively show that the three yeast acidic proteins are coded by independent genes and are not the result of a post-translational modification of the product of a unique gene, as in bacteria. Like most ribosomal protein genes, the gene for protein L44' has an intron and two upstream stimulatory boxes (UASrpg) fitting closely to the consensus sequence. The genes coding for proteins L44 and L45 lack introns and seem also exceptional in other characteristics of their sequences. Proteins L44 and L45 have amino acid sequences with about 80% similarity. Protein L44' is only 63% similar to the other two polypeptides. The three proteins have highly conserved carboxyl termini comprising the last 30 amino acids, and the first 10 amino acids of L44 and L45 are identical. The results cast doubts about the possibility of a similar role for the different acidic ribosomal proteins.
Highlights
From the Centro deBiologia Molecular, Consejo Superior de Investigaciones Cientificasand Uniuersidad Autonoma, Canto Blanco, 20849 Madrid, Spain
A unique copy of each gene is detected using the phage cDNA inserts as probes in nitrocellulose blots of yeast DNA digested with different restricribosomal protein L10, forming a characteristic protuberance of the large ribosomal subunit (see Moller and Maasen (1986) and Trautet al. (1986) for recent reviews)
The proas probes ingenomic DNA banks, DNA fragments car- teins have been called P1 and P2, and their sequences are rying theacidic protein genes havbeeen cloned, char- known in the case of rat and human livers either by protein acterized,and sequenced
Summary
Physical Characterization of cDNA Inserts Codingfor Acidic Proteins-By screening a cDNA librarypreparedwith poly(A)+mRNA from yeast in Xgtll with specific polyclonal and monoclonal antibodies (see Miniprint), three recombinant phages, XgtB242, XgtBl71, and XgtB302, which express proteins L45, L44, and L44’, respectively, were obtained. Gene CopyNumber forAcidic Proteins-The cDNA inserts were used as probes in Southern blots of total yeast DNA digested with HindIII and HincII (Fig. 2). A 5.9-kb3DNA fragment was detected in the positive clones, from which a 3.7-kb EcoRI piece containing the insert 242 hybridizing part was subcloned into plasmid pUC18. To assure the cloning of the L44’gene, a minibank was constructed by inserting yeast DNA fragments hybridizing with insert 302 into the HindIII restriction site of plasmid pUC18. The restriction enzyme maps of the genomic DNA inserts subcloned into pUC18 were determined (Fig. 3). Part of the inserts, including the cDNA hybridizing fragment and its 3‘- and 5”flanking regions, was sequenced by the dideoxy chain termination method using complementary synthetic oligonucleotides as primers 49 SVDELITEGNEKLAAVP..AAGPASACGAAAASCDAAAEEEKEEEAAEES 96 flanking regions show several interesting characteristics
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