Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits.

QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.

SQT1, which encodes an essential WD domain protein of Saccharomyces cerevisiae, suppresses dominant-negative mutations of the ribosomal protein gene QSR1.

QSR1 is an essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein required for joining of 40S and 60S subunits. Truncations of QSR1 predicted to encode C-terminally truncated forms of Qsr1p do not substitute for QSR1 but do act as dominant negative mutations, inhibiting the growth of yeast when expressed from an inducible promoter. The dominant negative mutants exhibit a polysome profile characterized by 'half-mer' polysomes, indicative of a subunit joining defect like that seen in other qsr1 mutants (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997.) By screening a high-copy yeast genomic library, we isolated several clones containing overlapping inserts of a novel gene that rescues the slow-growth phenotype of the dominant negative qsr1 truncations. The suppressor of qsr1 truncation mutants, SQT1, is an essential gene, which encodes a 47.1-kDa protein containing multiple WD repeats and which interacts strongly with Qsr1p in a yeast two-hybrid system. SQT1 restores growth and the "half-mer" polysome profile of the dominant negative qsr1 mutants to normal, but it does not rescue temperature-sensitive qsr1 mutants or the original qsr1-1 missense allele. In yeast cell lysates, Sqt1p fractionates as part of an oligomeric protein complex that is loosely associated with ribosomes but is distinct from known eukaryotic initiation factor complexes. Loss of SQT1 function by down regulation from an inducible promoter results in formation of half-mer polyribosomes and decreased Qsr1p levels on free 60S subunits. Sqt1p thus appears to be involved in a late step of 60S subunit assembly or modification in the cytoplasm.

Effect of testosterone on the growth properties and on epidermal growth factor receptor expression in the teratoma-derived tumorigenic cell line 1246-3A.

1246-3A is an insulin-independent tumorigenic cell line isolated from the C3H mouse teratoma-derived adipogenic cell line 1246. In the present paper, we have demonstrated that testosterone inhibits the in vivo tumorigenic properties of the 1246-3A cells. Castrated male mice receiving injections of 1246-3A cells developed larger tumors at a higher frequency than sham-operated animals. Administration of testosterone to castrated male mice resulted in a dramatic decrease in tumor development. In vitro studies indicated that testosterone inhibited by 50% the proliferation of the 1246-3A cells in culture. However, growth inhibition was observed only if the cells had been cultivated in the presence of testosterone for at least 4 days. In contrast, testosterone had little effect on the proliferation of the parent cell line 1246. Binding of several polypeptide growth factors was examined in cells cultivated in the absence and in the presence of testosterone. Testosterone increased 125I-EGF specific binding to 1246-3A cells. Scatchard analysis of EGF binding indicated that testosterone treatment induced a 2.4-fold increase in the number of cell surface EGF binding sites. This was accompanied by an increase in the intensity of cross-linked EGF receptors on the cells treated with testosterone. In addition, 1246-3A cells cultivated for 9 days in the presence of testosterone displayed a 10-fold increase in the level of EGF receptor mRNA when compared to 1246-3A cells maintained in its absence. Similar to its action on cell proliferation, the increase in EGF receptor number and mRNA expression was observed mainly if 1246-3A cells had been exposed to testosterone for 9 days. The data presented in this paper demonstrate that both in vivo and in vitro, testosterone induces in the teratoma-derived 1246-3A cell line phenotypic changes such as growth inhibition and modulation of EGF receptor expression.

The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene.

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.

Exchangeability of Qsr1p, a large ribosomal subunit protein required for subunit joining, suggests a novel translational regulatory mechanism.

Qsr1p is a 60S ribosomal subunit protein that is necessary for joining of large and small ribosomal subunits and is also one of the last proteins assembled onto the 60S ribosomal subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large ribosomal subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S ribosomal subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S subunits.

Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties.

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.

Structure of the gene encoding mouse adipose differentiation-related protein (ADRP).

Adipose differentiation-related protein (ADRP) is a novel 50-kDa membrane-associated protein whose message levels are induced rapidly and maximally after triggering adipocyte differentiation. The gene encoding mouse ADRP has been isolated and characterized from four overlapping lambda phage clones. The gene spans 14 kb and contains 8 exons and 7 introns. Exons range in size from 50 to 696 bp and intron sizes range from 87 bp to 4.3 kb. Major and minor transcription initiation sites were determined 76 and 78 bp, respectively, upstream of the initiator methionine. A TATTTTA sequence is centered 30 bp upstream of the major transcription start site, and within the 5'-flanking region there are several putative transcription factor binding sites. ADRP has been mapped to chromosome 4, specifically between the b and Ifa loci. A second ADRP-like gene was isolated and partially characterized. This second locus is not expressed in 12 different mouse tissues and shares 87% sequence similarity to ADRP over exon and intron regions analyzed. Finally, this is the first reported genomic structure of ADRP.

A novel mouse gene highly conserved throughout evolution: regulation in adipocyte differentiation and in tumorigenic cell lines.

A cDNA clone referred to as 168 was previously isolated from mouse 1246 adipocytes by differential hybridization on the basis of its down regulation in adipocytes when compared to preadipocytes. 5' RACE was used to obtain a full length clone of 761 bp encoding for a highly basic 25 kD polypeptide that is extremely conserved in several diverse species of eukaryotes. There is a single amino acid substitution at position 202 compared to the human homolog, QM, a putative tumor suppressor. Clone 168 mRNA decreases 80% in rat primary culture of adipocytes compared to preadipocytes and does not decrease when differentiation is blocked by PGF2 alpha or EGF, indicating that the decrease is correlated with expression of the differentiation phenotype. Finally, two 1246 cell line variants that exhibit altered growth and increased tumorigenicity have a similar level of 168 mRNA when compared to the non tumorigenic adipogenic parent cell line.