Differential subcellular localization of ribosomal protein L7 paralogs in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In Saccharomyces cerevisiae, ribosomal protein L7, one of the ∼46 ribosomal proteins of the 60S subunit, is encoded by paralogous RPL7A and RPL7B genes. The amino acid sequence identity between Rpl7a and Rpl7b is 97 percent; they differ by only 5 amino acid residues. Interestingly, despite the high sequence homology, Rpl7b is detected in both the cytoplasm and the nucleolus, whereas Rpl7a is detected exclusively in the cytoplasm. A site-directed mutagenesis experiment revealed that the change in the amino acid sequence of Rpl7b does not influence its sub-cellular localization. In addition, introns of RPL7A and RPL7B did not affect the subcellular localization of Rpl7a and Rpl7b. Remarkably, Rpl7b was detected exclusively in the cytoplasm in rpl7a knockout mutant, and overexpression of Rpl7a resulted in its accumulation in the nucleolus, indicating that the subcellular localization of Rpl7a and Rpl7b is influenced by the intracellular level of Rpl7a. Rpl7b showed a wide range of localization patterns, from exclusively cytoplasmic to exclusively nucleolar, in knock-out mutants for some rRNA-processing factors, nuclear pore proteins, and large ribosomal subunit assembly factors. Rpl7a, however, was detected exclusively in the cytoplasm in these mutants. Taken together, these results suggest that although Rpl7a and Rpl7b are paralogous and functionally replaceable with each other, their precise physiological roles may not be identical.     

Cloning and characterization of a ribosomal protein L23a from Haemaphysalis qinghaiensis eggs by immuno screening of a cDNA expression library

A primary cDNA library with a size of 1.34 × 10⁶ PFU was constructed from Haemaphysalis qinghaiensis eggs and was immunoscreened with rabbit anti-H. qinghaiensis serum. One clone (Hq22, named following those clones obtained from adult Haemaphysalis qinghaiensis cDNA library which we constructed before) screened from the cDNA library was selected randomly for sequencing. The entire sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). A search of the cloned sequence against GenBank revealed that it related to ribosomal protein L23a (Rpl23a) and had a high percentage similarity to this protein from different species. Conserved domains for Rpl23a were also identified in the cloned sequence. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other tissues and different developmental stages of H. qinghaiensis. Based on the H. qinghaiensis Rpl23a sequence, open reading frames (ORF) of Rpl23a of Heamaphysalis longicornis and Boophilus microplus were also cloned and were performed for comparison with Rpl23a of H. qinghaiensis and other organisms as well. Vaccine based on Rpl23a recombinant protein cannot protect sheep against H. qinghaiensis.     

Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Δrpl31aΔrpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Δrpl31aΔrpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Δrpl31aΔrpl31bΔzuo1 did not exceed that of Δrpl31aΔrpl31b or Δzuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Δrpl31aΔrpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.     

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast

A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

Studies on Rabies Virus RNA Polymerase: 1. cDNA Cloning of the Catalytic Subunit (L Protein) of Avirulent HEP-Flury Strain and Its Expression in Animal Cells

To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP-Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA-transfected COS-7 cells displayed almost the same electrophoretic mobility as that of authentic L protein. Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body-like granular distribution, suggesting that the inclusion body-like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.