Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis

LY171883, a peroxisome proliferator and leukotriene D4-antagonist, induced a statistically significant increase in the number of hepatic lesions in B6C3F1 female mice in a 2 year oncogenicity study at dietary doses of 0.0225% and 0.075%. The mutation frequency and spectrum of the 61st codon of H-ras was determined for 64 independent, archived lesions from the LY171883 2 year oncogenicity study using the polymerase chain reaction (PCR), allele specific oligo hybridization (ASO) and DNA sequencing. Results showed 41 (64%) of these lesions had mutations at the 61st codon (16/21 hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26 focal hepatocellular hyperplasias and 2/7 focal hepatocellular atypia). These mutations consisted of 18 C-A transversions, 16 A-G transitions and seven A-T transversions. Compared to the mutation frequency for spontaneously occurring archival B6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions (64%) was significantly higher (P < 0.01). The frequencies of H-ras 61st codon mutations among the LY171883 lesion types (hepatocellular carcinomas 76%, hepatocellular adenomas 40%, focal hepatocellular hyperplasias 73% and hepatocellular atypia 29%) were also significantly different (P = 0.035). In contrast, spontaneous lesions showed no statistical difference in the frequencies of mutation among lesion types (P > 0.5). The mutation spectrum of the LY171883 lesions was not significantly different from the spontaneous spectra. It may be concluded that based on the similarity in mutation spectrum and the increase in mutation frequency, LY171883 may selectively promote spontaneous hepatic lesions containing H-ras 61st codon mutations. In addition, the difference in mutation frequency among lesion types does not support a linear progression of all LY171883 lesions through focal atypia, focal hepatocellular hyperplasias, hepatocellular adenomas and hepatocellular carcinomas.     

Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein

We determined the genomic organization of human CRF type-1 receptor (hCRF-R1). The gene coding for hCRF-R1 consists of at least 14 exons and spans over 20 kilobases. hCRF-R1's three reported isoforms originate from the same gene by alternative splicing. The first hCRF-R1, which binds to CRF with the highest affinity and transduces the most sensitive cAMP accumulation in response to CRF, is encoded in a total of 13 exons, the only one excluded being exon 6. The second isoform contains an additional 29-amino acid sequence which corresponds to exon 6. Unlike the first isoform, the third lacks a 40-amino acid sequence, corresponding to exon 3. Exon-intron boundaries are the same as that of the consensus sequence. Locations of introns in the coding sequence are similar to human CRF-R1, rat CRF-R1, human CRF-R2alpha and others belonging to the human glucagon receptor family.     

Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression

Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10. To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human FGF-10 and 91% identical to rat FGF-10. When expressed in mammalian 293 cells, the mFGF-10 protein was glycosylated but remained cell- or extracellular matrix-associated. Upon addition of heparin, mFGF-10 protein was released into the media. mRNA encoding mFGF-10 was relatively abundant in lung, skin, brain and heart. In the skin, both FGF-7 and mFGF-10 were expressed in the dermal, but not the epidermal compartment. In contrast to FGF-7, mFGF-10 expression was not induced during cutaneous wound repair. In cultured fibroblasts, expression of mFGF-10 was strongly repressed by transforming growth factor beta and tumor necrosis factor alpha, whereas epidermal growth factor and interleukin-1beta had no effect. These results demonstrate a differential regulation of mFGF-10 and FGF-7 expression in vitro and during the wound healing process.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5'- and 3' untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3' untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5' untranslated regions, but relatively little homology of the 5' untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3' untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.     

Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.     

Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPgammaS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3'-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3' termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.     

A monocyte chemotactic factor, S19 ribosomal protein dimer, in phagocytic clearance of apoptotic cells

HL-60 cells derived from a human promyelocytic leukemia underwent apoptosis by heat treatment. When the heat-treated HL-60 cells were injected into guinea pig skin, monocyte/macrophage infiltration was observed 24 or 36 hours later, and the apoptotic cells were phagocytically cleared by 48 hours after their injection. The infiltration and clearance patterns were quite different from those observed in injection of necrotic or boil-fixed HL-60 cells. The apoptotic cells released a monocyte chemotactic factor in vitro 24 hours after the heat treatment. The chemotactic factor generated was identified as the cross-linked homodimer of S19 ribosomal protein by its immunologic and physicochemical properties. A serine protease that inactivates the monocyte chemotactic factor was also released from the apoptotic cells 30 hours after the heat treatment. A super infusion of this protease into the skin where the apoptotic cells had been injected diminished the number of infiltrated monocytes. The present results indicate an important role of the S19 ribosomal protein dimer in the phagocytic clearance of apoptotic cells.     

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 40 S ribosomal subunit proteins Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29

The proteins of the small subunit of rat liver ribosomes were separated into five main groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Twenty-one proteins (Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29) were isolated from three groups (A40, C40, and D40) by ion exchange chromatography on DEAE-cellulose, carboxymethylcellulose, and phosphocellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.1 to 11 mg. Six of the proteins (S5', S10, S11, S18, S19, and S27') had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Isolation and characterization of the mitochondrial ATP synthase from Chlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the alpha subunit

We have isolated the F0F1-ATP synthase complex from oligomycin-sensitive mitochondria of the green alga Chlamydomonas reinhardtii. A pure and active ATP synthase was obtained by means of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and PMSF [corrected]. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase alpha subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58-70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase beta subunit from C. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771-780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15-18 residues longer than in ATP synthase alpha subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.