Umbilical separation time delayed by alcohol application.

AIMS: To compare the effects on time of umbilical cord separation of cleaning with 95% alcohol and natural drying in a high-humidity subtropical country. METHODS: One hundred and fifty neonates were randomly assigned to two groups, 75 in each. For the control group, umbilical cleansing with 95% alcohol was performed after daily bathing; natural drying without a topical regimen was used for the trial group. RESULTS: Complete information was obtained for 71 neonates in the control group and 71 in the trial group. At 1 month after delivery, no enrolled neonate had developed omphalitis or skin infection. Cord separation time was significantly reduced for the natural-drying group compared with the alcohol-cleansing group (p=0.014). In both groups, separation time was longer for newborns delivered by caesarean section than for those delivered vaginally (p=0.001). Nine mothers in the trial group and five in the control group complained of discharge from the umbilicus. Separation time was not influenced by gender, gestational age, birthweight or length, gravidity, meconium staining, maternal age or presence of discharge. CONCLUSIONS: Cleaning with 95% alcohol did not reduce umbilical cord separation time. This traditional method is not necessary for routine cord management, even in a subtropical country.

Development of amoxicillin enzyme-linked immunosorbent assay and measurements of tissue amoxicillin concentrations in a pigeon microdialysis model.

A sensitive ELISA was developed for the detection of amoxicillin (AMX) in serum, urine, and milk. The ELISA used an indirect competitive method produced by coating the plate with ovalbumin conjugated with AMX hapten. Antibodies against AMX-BSA were detected by a goat-antirabbit antibody conjugated with peroxidase. Calibration standard curves ranged from 1.28 ng/mL to 20 microg/mL [IC(50) (inhibition concentration 50%) = 100 ng/mL], and the limits of detection were 1.3, 2.7, and 4.8 ng/mL for urine, milk, and serum, respectively. The intra- and interassay variations were less than 4 and 9.6%. The antibody produced against AMX cross-reacted highly with penicillin G (77%); cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.9, 51.4, and 48.8%, respectively); but was considered non-cross-reactive with dicloxacillin (7.4%), cefadroxil (<1%), and cefazolin (<1%). Concentrations of AMX were measured simultaneously in venous blood and muscles by using the developed AMX ELISA in an in vivo microdialysis model designed for pigeons. Following i.m. injection (25 mg/kg), AMX attained a peak blood level of 4.74 +/-0.30 mu g/mL and decreased with a half-life of 2.38 +/-0.16 h. In contrast, measurements in pectoral and femoral muscles exhibited delayed appearances, reduced peak concentrations, and prolonged half-lives of 4.07 +/-0.48 (pectoral) and 3.01 +/-0.26 (femoral) that were significantly different from each other and those in the blood (P < 0.05). Blood protein binding was calculated to be 27.9 +/-5.7%. This study demonstrated the semiquantitative application of a selective AMX ELISA in the first microdialysis procedure for continuous monitoring of drug levels in specific tissues of pigeons and maybe useful for related studies in other poultry species.

Factors Determining Physical and Mental Quality of Life of Living Kidney Donors in Taiwan.

BACKGROUND: Living-donor kidney transplantation has a positive influence on recipients' life expectancy and improves quality of life for patients with end-stage renal disease compared with dialysis patients. Evaluation of the physical and mental quality of life for donors can promote positive perceptions about donation and help potential donors in their decision-making process. The aim of this study was to explore the predictive factors of quality of life for living kidney donors. METHODS: A cross-sectional and descriptive design was used, and the study was conducted from January to July 2013. The donors were a convenience sample of 34 participants who had undergone kidney transplant surgery >1 year earlier. RESULTS: The results showed that kidney donors had a low to moderate physical and mental quality of life. Multiple regression analysis revealed that financial concerns and anxiety explained 27.8% of the total variance of quality of life in the physical component. Anxiety and paid work explained 61.4% of the total variance of quality of life in the mental component. CONCLUSIONS: After renal transplantation, living kidney donors experienced low to moderate quality of life. Because donors are family members (siblings, sons or daughters, spouses, or parents), monthly family income is a significant issue that influences both the decision to donate and quality of life after transplantation. Our findings suggest that pre-transplantation assessment must include social workers as part of the health care team to evaluate the impact of a donor's financial status on post-transplantation quality of life.

Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA.

In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit. L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay. In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture. Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one. Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner. Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA. However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule. L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4x10(6)/M. L17 bound the pre-rRNA molecule preferentially (Ka=17x10(6)/M) but also bound the mature 25S rRNA species (Ka=10x10(6)/M). L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well (Ka=11-12x10(6)/M). L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with Ka=3.1 vs. 2.5x10(6)M, respectively. We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.

A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine.

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function.

Involvement of lysine 270 and lysine 271 of yeast 5S rRNA binding protein in RNA binding and ribosome assembly.

Contributions of the highly conserved K270 and its neighboring K271 in the C-terminal region of the yeast ribosomal protein L1 to 5S rRNA binding and ribosome assembly were examined by in vivo and in vitro studies on the consequences of 14 substitution mutations. All mutant proteins with a single amino-acid substitution at either position were able to bind 5S rRNA in vitro to an extent comparable to the wild-type. Yeast cells expressing these mutant proteins, except the K270G mutant, grew at nearly normal rates. Mutations of K270 appeared to produce more demonstrable effects than those of K271. The double mutant K270,271G bound RNA poorly and yeast cells expressing the mutant protein grew 30% slower. Double mutants K270,271E and K270,271R were lethal, although the mutant protein was assembled into the 60S ribosomal subunits. The resultant subunits were not stable leading eventually to cell death. The in vitro RNA binding ability of the respective protein was reduced by 60% and 20%. Taken together, the present data identified K270 and K271 as important amino-acid residues in the function of the yeast ribosomal protein L1.

Higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA synthesized in vitro.

Dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene-sulfonate, RNase T1 and RNase V1 have been used as structure-sensitive probes to examine the higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA molecule. Data produced have been used to evaluate several theoretical structure models for the 5.8 S rRNA sequence within the precursor rRNA. These models are generated by minimum free energy calculations. A model is proposed that accommodates 83% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. Several alternative suboptimal secondary structures have been evaluated. Moreover, the chemical reactivities of several residues within the 5.8 S rRNA sequence in the precursor rRNA molecule differ from those of the corresponding residues in the mature rRNA molecule. This finding provides experimental evidence to support the notion that the 5.8 S rRNA sequence within the precursor rRNA undergoes structural reorganization following rRNA processing.

Contributions of multiple basic amino acids in the C-terminal region of yeast ribosomal protein L1 to 5 S rRNA binding and 60 S ribosome stability.

Previous studies suggest that the C-terminal region of ribosomal protein L1 from Saccharomyces cerevisiae is important for its interaction with the 5 S rRNA molecule. Within this region are several highly conserved basic amino acids including Lys276, Lys279, Lys289, Arg282, Arg285. To examine potential contributions of these amino acids to RNA-protein interaction and ribosomal assembly, effects of substitutions of these residues by methionine either individually or in combinations were examined. A methionine substitution of any one of the lysine residues did not significantly affect RNA binding in vitro. The mutant RNPs were as stable as the wild-type RNP. Yeast transformants expressing these mutant proteins grew at the same rate as the wild-type. However, mutant proteins containing substitutions of any two of these basic amino acids bound RNA weakly. The resultant RNPs were significantly less stable than the wild-type. Whereas cells expressing mutant L1 with a single substitution at 289 was not lethal, cells expressing mutant L1 with any double substitutions involving Lys289 as one of the substituted amino acids were lethal. These data suggest that Lys289 plays a key role in the binding of ribosomal protein L1 to 5 S rRNA. The other basic residues, particularly Arg282, and Arg285, in this region also contribute to RNA binding. These residues are predicted to locate on the same side of an alpha helix. We would like to propose a structural model for the yeast RNP that involves multiple contact sites located on one side of the helix in the C terminus of the protein and the 5 S rRNA. These basic amino acids also participate, directly or indirectly, in the interaction of the RNP complex with other components of the 60 S ribosomal subunit.

Structural analysis of the internal transcribed spacer 2 of the precursor ribosomal RNA from Saccharomyces cerevisiae.

Full-length precursor ribosomal RNA molecules (6440 bases) were produced in vitro using a plasmid containing the yeast 35 S pre-rRNA operon under the control of phage T7 promoter. The higher-order structure of the internal transcribed spacer 2 (ITS-2) region (between the 5.8 S and 25 S rRNA sequence) in the pre-rRNA molecule was investigated using a combination of enzymatic and chemical structural probes. The data were used to evaluate several structural models predicted by a minimum free-energy calculation. The results supported a model in which the 3' end of the 5.8 S rRNA and the 5' end of the 25 S rRNA are hydrogen-bonded better than the one in which the ends are not. The model contains a high degree of secondary structure with several stable hairpins. Similar structural models for the ITS-2 regions of Schizosaccharomyces pombe, Saccharomyces carlsbergensis, mung bean and Xenopus laevis were derived. Certain common folding features appear to be conserved, in spite of extensive sequence divergence. The yeast model should be useful as a prototype in future investigations of the structure, function and processing of pre-rRNA.

Structure analysis of the 5' external transcribed spacer of the precursor ribosomal RNA from Saccharomyces cerevisiae.

Full-length precursor ribosomal RNA molecules were produced in vitro using as a template, a plasmid containing the yeast 35 S pre-rRNA gene under the control of the phage T3 promoter. The higher-order structure of the 5'-external transcribed spacer (5' ETS) sequence in the 35S pre-rRNA molecule was studied using dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, RNase T1 and RNase V1 as structure-sensitive probes. Modified residues were detected by primer extension. Data produced were used to evaluate several theoretical structure models predicted by minimum free-energy calculations. A model for the entire 5'ETS region is proposed that accommodates 82% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. The model contains a high degree of secondary structure with ten stable hairpins of varying lengths and stabilities. The hairpins are composed of the Watson-Crick A.T and G.C pairs plus the non-canonical G.U pairs. Based on a comparative analysis of the 5' ETS sequence from Saccharomyces cerevisiae and Schizosaccharomyces pombe, most of the base-paired regions in the proposed model appear to be phylogenetically supported. The two sites previously shown to be crosslinked to U3 snRNA as well as the previously proposed recognition site for processing and one of the early processing site (based on sequence homology to the vertebrate ETS cleavage site) are located in single-stranded regions in the model. The present folding model for the 5' ETS in the 35 S pre-rRNA molecule should be useful in the investigations of the structure, function and processing of pre-rRNA.

Inhibition of BMP receptor synthesis by antisense oligonucleotides attenuates OP-1 action in primary cultures of fetal rat calvaria cells.

Osteogenic protein-1 (OP-1 or bone morphogenetic protein-7 [BMP-7]) stimulates osteoblast differentiation in vitro and induces bone formation in vivo. BMPs exert their effects through complex formation with a heterodimeric receptor composed of a type I and a type II polypeptide. In the present study, mRNAs for three BMP subtype I receptors (ActR-I, BMPR-IA, and BMPR-IB) and one BMPR-II receptor were detected by Northern analysis in two human osteosarcoma cell lines (SaOS-2 and TE85) and in the primary cultures of fetal rat calvaria (FRC) cells. OP-1 affected the steady-state mRNA levels of these receptors differently among these cell types. To study the role of each receptor type in OP-1 action in FRC cells, receptor synthesis was inhibited by antisense oligonucleotides. Inhibition of receptor synthesis was confirmed by immunoprecipitation of radiolabeled cellular proteins with specific antibodies. The osteogenic action of OP-1 was measured by alkaline phosphatase (ALP) activity and mineralized bone nodule formation in FRC cells. Results showed that inhibition of synthesis of a single subtype I receptor alone did not affect significantly the OP-1-stimulated ALP activity. Inhibition of BMPR-II synthesis reduced the OP-1-stimulated ALP activity by about 50%. Inhibition of synthesis of any one of the type I receptor plus the BMPR-II receptor did not reduce the OP-1-stimulated ALP activity significantly beyond that observed by inhibition of BMPR-II alone. Under these conditions, nodule formation was affected similarly, thus supporting the observations made with the ALP measurements. The present results suggest that the ActR-I, BMPR-IA, and BMPR-IB receptors and the BMPR-II receptor are expressed and functional for OP-1 in FRC cells and that regulation of synthesis of these receptors may be a mechanism by which a specific cell type responds to OP-1. The turnover rate of these receptor proteins might be relatively long and another type II receptor(s) for OP-1 might be functional in FRC cells.

Identification of an osteogenic protein-1 (bone morphogenetic protein-7)-responsive element in the promoter of the rat insulin-like growth factor-binding protein-5 gene.

Osteogenic protein-1 (OP-1), a member of the bone morphogenetic protein subfamily of the transforming growth factor-beta superfamily, induces new bone formation in vivo and regulates the expression of numerous growth factors. We previously showed that OP-1 down-regulates the transcription of the insulin-like growth factor-binding protein-5 (IGFBP-5) in primary cultures of fetal rat calvaria (FRC) cells. In the present study we identified, within the IGFBP-5 promoter, a 21-bp region that confers OP-1 responsiveness in FRC cells. Within this region lie three putative cis-acting regulatory elements, viz. a CAAT-like sequence, a CCAAT/enhancer-binding protein (C/EBPalpha)-like element, and a c-Myb or E-box-like motif. Mutations in the CAAT-like sequence reduced the promoter activity in both control and OP-1-treated cells, but did not abrogate the OP-1-induced down-regulation. Mutations in the C/EBPalpha-like element reduced the promoter activity in both control and OP-1-treated cells without significantly affecting the extent of down-regulation. Mutations in the putative c-Myb or E-box-like motif reduced the promoter activity in both the OP-1-treated and control cells and completely abolished the inhibitory effect of OP-1 on the IGFBP-5 promoter activity. Gel mobility shift analyses further showed specific interaction between nuclear protein(s) in FRC cells and the 21-bp region. OP-1 down-regulates the nuclear regulatory protein interaction with the 21-bp region by reducing either the cellular concentration of the regulatory protein(s) or the affinity of the regulatory protein(s) for the OP-1 responsive element. In conclusion, we identified an OP-1 response region in the rat IGFBP-5 promoter and further showed that OP-1 down-regulates the nuclear protein interaction with the response element(s).

Differential regulation of IGF-binding protein gene expression by cAMP in rat C6 glioma cells.

We previously reported that cAMP inhibits autocrine IGF-I gene expression in rat C6 glioma cells. In this study we examined the influence of cAMP on IGF-binding protein gene expression in C6 cells. cAMP potently inhibited IGF-binding protein-3 mRNA and, to a lesser extent, IGF-binding protein-4 mRNA and transiently stimulated IGF-binding protein-5 mRNA. The changes in secreted IGF-binding proteins whose molecular weights were consistent with IGF-binding protein-3 and -5 correlated with those of mRNA levels. cAMP decreased the IGF-binding protein-3 mRNA half-life, but did not alter IGF-binding protein-4 and -5 mRNA half-lives. An IGF-binding protein-5 promoter/luciferase fusion construct containing 888 bp of 5'-flanking sequence and the first 114 bp of exon 1 sequence was stimulated by cAMP after 24 h by approximately 2-fold in transient transfection assays. 5'- or 3'-deletion to -33 or +10 (the transcription start site was designated as +1), respectively, did not alter the increase caused by cAMP. Site-directed mutagenesis of the region from -14 to -5 led to a loss of the ability of the IGF-binding protein-5 promoter to respond to cAMP. H89, a cell-permeable protein kinase A inhibitor, did not alter the regulation of IGF-binding protein mRNAs in response to cAMP.

Osteogenic protein-1-mediated insulin-like growth factor gene expression in primary cultures of rat osteoblastic cells.

Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein family and has been shown to induce new bone formation in vivo. In the present study, we determined whether the expression of the IGF system, a significant growth factor system in bone, was altered by OP-1 in primary cultures of fetal rat calvarial cells. Levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, IGF-I receptor, and IGF-binding proteins (IGFBP-1 to -6) were determined after OP-1 treatment. The level of total IGF-I mRNA was elevated in an OP-1 concentration (0-1000 ng/ml)-dependent manner, with maximal stimulation of IGF-I mRNA of 2- to 3-fold apparent 24 h after treatment. The increase in the IGF-I mRNA level involved a preferential stimulation of transcripts initiated at start site 2 in the exon 1 promoter. The level of IGF-II mRNA also increased by approximately 2-fold in OP-1 treated cells in a concentration-dependent manner. The level of IGF-I receptor mRNA was not altered by treatment. Whereas IGFBP-1 mRNA was not detected in these cells, IGFBP-2 mRNA was expressed, but the expression was not changed after treatment for 48 h in the concentration range (0-1000 ng/ml) tested. The IGFBP-3 mRNA level was increased slightly 48 h after OP-1 treatment in a concentration-dependent manner. The IGFBP-4, -5, and -6 mRNA levels decreased dramatically in an OP-1 concentration-dependent manner. In addition, coincubation of antisense oligonucleotides corresponding to IGF-I or -II mRNA sequence with OP-1 reduced the OP-1 induced elevation in alkaline phosphatase activity. The present results suggest that the differentiation of rat osteoblastic cells in response to OP-1 is mediated in part by increased IGF-I -II gene expression and alterations in the gene expression of different IGFBPs.

Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation.

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) alters the steady state levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) in primary cultures of fetal rat calvaria (FRC) cells. In the present study, the effects of exogenous IGF-I on bone cell differentiation and mineralized bone nodule formation induced by OP-1 were examined. Exogenous IGF-I synergistically and dose dependently enhanced OP-1 action in stimulating [3H]thymidine incorporation, alkaline phosphatase activity, PTH-dependent cAMP level, and bone nodule formation. Maximal synergism between OP-1 and IGF-I was observed when both factors were added simultaneously. Synergism was not observed when FRC cells were pretreated with IGF-I for 24 h, followed by OP-1 treatment. These findings suggest that IGF-I acted on OP-1-sensitized FRC cells. To examine the mechanism(s) by which this sensitization may occur, levels of mRNA encoding OP-1 receptor, IGF-I receptor, and IGFBPs were measured. The mRNA levels of both type I and II OP-1 receptors were elevated by OP-1, but were not changed further by combined OP-1 and IGF-I treatment. IGF-I receptor gene expression was not changed by OP-1, IGF-I, or a combination of both factors. OP-1 alone or together with IGF-I increased the steady state IGFBP-3 mRNA level and reduced the steady state mRNA levels of IGFBP-4, -5, and -6. IGF-I alone did not change the steady state mRNA levels of IGFBP-3, -4, and -6, but elevated that of IGFBP-5. Des(1-3)-IGF-I, which has a lower affinity for IGFBPs, was more effective than the full-length IGF-I in enhancing the OP-1-induced alkaline phosphatase activity. Exogenous IGFBP-5 inhibited the OP-1-induced alkaline phosphatase activity and reduced the synergistic stimulatory effect of IGF-I and OP-1. These findings strongly suggest that the OP-1-induced down-regulation of IGFBPs, especially that of IGFBP-5, is an important mechanism by which OP-1 and IGF-I synergize to stimulate FRC cell differentiation.

Molecular cloning of cDNA for human prostatic acid phosphatase.

A human liver cDNA library in lambda gt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, lambda Hap21 and lambda Hap22 were further characterized: clone lambda Hap21 contained a 0.8-kb cDNA insert and clone lambda Hap22 a 1.8-2.0-kb insert. XbaI digestion of lambda Hap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone lambda Hap22 contained all the genes carried by lambda gt11(lac5cI857nin5Sam100) and the 2-kb insert. An Escherichia coli(lambda Hap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(lambda Hap22) lysate revealed that the non-induced lambda Hap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-beta-galactosidase and was produced only upon induction with IPTG. These results indicated that lambda Hap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.

An in vitro system for studying RNA-protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA.

Previous attempts to study the binding of yeast ribosomal protein L1 with 5S rRNA in vitro have been impeded by the failure to form RNA-protein complexes with purified protein and RNA. To circumvent this difficulty, we have developed an in vitro system that allowed RNP formation. The system involved in vitro expression of the protein L1 from its cloned gene in the presence of exogenous yeast 5S rRNA. A protein of the expected size (34 kDa) was synthesized by in vitro transcription and translation. A specific 5S rRNA-protein L1 complex (RNP) was formed when the rRNA molecule was present during protein L1 synthesis. However, the full-length protein L1 failed to bind 5S rRNA. The extent of RNP formation was proportional to the concentration of the exogenous yeast 5S rRNA in the reaction. The RNP displayed properties identical to those isolated from mature 60S ribosome subunits. Addition of yeast 5.8S rRNA did not result in the formation of a specific RNP. Using this in vitro system, we examined the ability of several deletion mutant proteins to bind yeast 5S rRNA and concluded that protein L1 missing residues 261 to 295 from the C-terminus could not bind yeast 5S rRNA. This in vitro system should be useful for future studies on the molecular nature of 5S rRNA-protein L1 interaction.

In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein.

Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable.

The initiation codon AUG binds at a hydrophobic site on yeast 40S ribosomal subunits as revealed by fluorescence studies with bis (1,8-anilinonaphthalenesulfonate).

Binding studies of yeast 40S ribosome with bis (1,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 microM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobic site on the yeast 40S subunit.

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.