Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Biosynthesis of carnitine in Neurospora crassa.

In growing cultures of Neurospora crassa lysine auxotroph 33933, (a) beta-hydroxy-epsilon-N-trimethyllysine and gamma-N-trimethylaminobutyraldehyde, postulated precursors of carnitine in the rat, effectively blocked synthesis of labeles carnitine from epsilon-N-[CH3-3H]trimethyllysine; and (b) beta-hydroxy-epsilon-N[CH3-3H[trimethyllysine and gamma-N-[CH3-3H]trimethylaminobutyraldehyde were effectively utilized for carnitine formation. From these isotopic experiments, the latter steps of carnitine synthesis in Neurospora are postulated to be epsilon-N-trimethyllysine leads to beta-hydroxy-epsilon-N-trimethyllysine leads to gamma-N-trimethylaminobutyraldehyde leads to gamma-butyrobetaine leads to carnitine.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.     

Specificity of nucleoside transport in Neurospora crassa.

The specificity of nucleoside uptake in germinating conidia of Neurospora crassa was investigated by examining the kinetics of [2-14C]uridine and [8-14C]-adenosine uptake in the wild-type, ad-8, and ud-1 pyr-1 strains. The results obtained strongly indicate that nucleoside transport in N. crassa is mediated solely by a general transport system which accepts both purine and pyrimidine nucleosides. Studies directed at characterizing the specificity of the transport system indicate that general structural features of the nucleoside which enhance its efficiency in binding to the transport system include: (i) a purine or pyrimidine as the heterocyclic ring, (ii) an unfunctionalized ribose or 2'-deoxyribose as the sugar unit, (iii) a beta-configuration about the anomeric carbon, (iv) the absence of substituents at C8 in the purine series and at C5 and C6 in the pyrimidine series, (v) the presence of a C5-C6 double bond in the pyrimidine series, and (vi) the absence of a charge on the heterocyclic ring.     

Utilization of labelled uridine, cytidine and orotic acid for determination of ribonucleic acid synthesis in mouse liver

[3H]uridine and [3H]orotic acid were equally utilized for labelling of RNA in mouse liver. Incorporation of [3H]cytidine was 2-3 times as high as that of [3H]-labelled uridine or orotic acid. These results differ from findings in rat liver, where both cytidine and orotic acid are better utilized for RNA labelling than is uridine. The ratio between liver RNA [3H]-activity and volatile [3H]-activity was 2, 3 and 13, respectively, at 300 min after injection of labelled uridine, orotic acid and cytidine, indicating an efficient chanelling of cytidine into liver anabolic pathways.     

Synthesis of nucleosides labeled with tritium at the 5'-carbon atom of the ribose residue

Chemical and enzymatic syntheses of [5'-3H]adenosine, [5'-3H]guanosine, and [5'-3H]uridine have been developed. The reduction of beta-D-ribo-pentadialdo-1,4-furanosyl derivatives of corresponding bases is used in the chemical synthesis. The maximum molar activity of the labelled products was 220 TBk/mol in reactions with [3H]NaBH4 and 370-740 TBk/mol in reactions with gaseous tritium. The enzymatic synthesis was performed by the rebosylation of heterocyclic bases with nucleoside phosphorylase and [5'-3H]uridine as a ribosyl donor. Nucleoside phosphorylase is proposed to be used in the immobilized form to avoid the decrease of molar activity. Nucleosides labelled with tritium both in ribosyl and heterocyclic moieties were synthesised enzymatically.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Metabolism of uridine and determination of liver ribonucleic acid synthesis in developing and adult mice

The metabolism of [5-3H]uridine and the incorporation of the precursor into liver RNA was studied in developing (13-day-old) and adult (45-day-old) mice. Different time-courses of labelling and increased amounts of labelled catabolic products of uridine were found in liver and blood of developing mice compared with adult animals. This is suggested to be a consequence of enlarged metabolite pools resulting from a lower total amount of uracil-degrading enzymes in the developing mice. The labelling of the uracil nucleotides was decreased in the developing liver. However, in spite of a lower specific radioactivity of UTP, the RNA-specific radioactivity of developing liver was increased compared with adult liver. Also the labelling of liver RNA with [6-14C]orotic acid was found to be increased in developing mice, thus indicating a higher rate of RNA synthesis in these animals. A more pronounced difference in liver RNA labelling between the developing and the adult mice obtained with the use of [14C]orotic acid than with [3H]uridine may suggest that the de novo pathway, relative to the salvage pathways, is more important in developing than in adult liver.     

A study of the heat-shock response in Neurospora crassa

1. Neurospora crassa was grown at 28 degrees C for 12 hr and transferred to higher temperatures for 2 hr. 2. Cultures labelled with [35S]methionine showed the synthesis of several new proteins in response to heat-shock at 46 to 48 degrees C. 3. Major polypeptides of approximate Mr 105,000, 99,000, 78,000, 43,000 and 23,000 were detectable in one-dimensional SDS-polyacrylamide slab gel electropherograms. 4. 2-D analysis using isoelectric-focussing in the first dimension and electrophoresis in SDS-polyacrylamide gels in the second led to the resolution of some of the heat-induced polypeptide into multiple spots differing in pI values. 5. mRNA from heat-shocked cells was translated poorly in Wheat Germ extract and rabbit reticulocyte lysate in vitro translation systems.     

Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine.

Neurospora crassa cytochrome C biosynthetically labelled with [S-Me-13C]methionine was prepared and analysed by 13C nuclear-magnetic-resonance spectroscopy. The methyl group of methionine is extensively incorporated into an N-trimethyl-lysine-72 residue arise from S-adenosylmethionine transmethylation, and that the methyl carbons of methionine residues are sufficiently close to the haem centre to experience chemical shifts from the ring currents of the tetrapyrrole pi electrons and broadening due to binding of methionine-80 with the haem, as well as interaction of the S-E113C]methyl groups with the paramagnetic iron centre. Although whole cells of the labelled Neurospora produced a 13C resonance at the expected position for the methionyl methyl group most of the methyl label was diverted into N-tetra-alkyl ammonium compounds. After an active state of growth these labelled N-methyl compounds appear, in the main, to be low-molecular-weight derivatives of choline which, if associated with membrane, are in a sufficiently fluid environment to have short rotational correlation times. During a subsequent dormant growth period these compounds become associated to some extent with relatively more immobile phases as a result of membrane binding or an increase in membrane rigidity.     

In vivo studies on the metabolism of pyrimidine nucleosides (author's transl)]

3H-labelled metabolites were determined in the perchloric acid-soluble fraction of blood plasma and liver of adult male Wistar rats, following the application of [5 - 3H]uridine. Ten minutes after the injection of uridine, only 20% of the total 3H activity of the plasma could be attributed to [3H]uridine. The remaining radioactivity was found chiefly in [3H]uracil (40%) and 3H2O (20%). In the liver, at 10 min, [3H]-uridine and [3H]uracil together accounted for less than 0.5% of the total radioactivity; about 70% of the radioactivity was due to [3H]beta-alanine, and 15% to 3H2O. 45 min after the injection, 70% of the radioactivity in the plasma was due to 3H2O, whereas uridine and uracil represented about 4% and 6%, respectively. At this time, about 55% of the radioactivity in the liver was due to [3H]beta-alanine, about 40% to 3H2O, and about 5% to unidentified metabolites; [3H]uridine and [3H]uracil were not observed. A comparison of the rate of catabolism of [5-3H]-uridine, [5-3H]cytidine and [6-3H]thymidine showed that cytidine is degraded in the organism 25 times more slowly than uridine or thymidine. The biological half lives for the total degradation of the [3H]nucleosides to 3H2O, based on the values in the plasma, were: uridine 1.1 h; thymidine 1.3 h; cytidine 25 h. Furthermore, the turnover time of exogenous uridine in the plasma was found to be 9 min, which gives a half life of 6 min for the metabolism of exogenous uridine to uracil.     

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.     

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.     

A missense mutation in the oxi-3 gene of the [mi-3] extranuclear mutant of Neurospora crassa

We have determined the DNA sequence of the oxi-3 gene and its 5' flanking region in the extranuclear [mi-3] mutant of Neurospora crassa. The oxi-3 gene encodes subunit 1 of cytochrome c oxidase, a protein known to be altered in the [mi-3] mutant (Bertrand, H., and Werner, S. (1979) Eur. J. Biochem. 98, 9-18). When the sequence from [mi-3] was compared to previously published sequences of the same region of mtDNA from wild-type N. crassa, a total of five differences was found. Four of these differences can be accounted for as either genetic polymorphisms or previous errors in DNA sequence determination. The remaining difference is a G/C to T/A transversion that changes a codon specifying an aspartic acid residue (GAC) to one that would specify tyrosine (TAC) at amino acid 448 of the 555 amino acid mature subunit 1 protein. This alteration was also found in the mtDNA of two separate heterokaryotic strains that had acquired the [mi-3] phenotype after repeated subculturing of heterokaryons forced between an [mi-3] strain and a strain containing a wild-type cytoplasm. The particular aspartic acid residue that would be affected by the mutation observed in [mi-3] is conserved in a diversity of species as either aspartic acid or glutamic acid, suggesting that an acidic residue at this position is important for the correct function of the subunit 1 protein. For these reasons, we consider it likely that the observed missense mutation is responsible for the [mi-3] phenotype.     

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Repair of ultraviolet light-induced damage to the deoxyribonucleic acid of Neurospora crassa

A method is described for labeling a specific pyrimidine in the deoxyribonucleic acid (DNA) of Neurospora crassa. In cells grown in the presence of [5-(3)H]-uridine, more than 97% of the radioactivity associated with the DNA had been incorporated into cytosine. The specific activity of the labeled DNA was approximately 3 x 10(3) counts per min per mug. The DNA was isolated by elution from hydroxyapatite columns with sodium phosphate buffer (0.40 m, pH 6.8). This procedure was used to demonstrate that in vegetative cells of N. crassa both photoreactivation and excision repair are operative, as measured by the removal of ultraviolet light-induced cytosine-containing dimers.     

Isolation and characterization of a DNA-uptake-stimulating protein from the culture medium of Neurospora crassa slime strain

A protein fraction was purified to homogeneity from the culture medium of the wall-less (slime) strain of Neurospora crassa (FGSC 1118), which proved to be identical with DNA-uptake-stimulating factor (designated DUSF), which has been described earlier [Schablik, M. and Szabó, G. (1981) FEMS Microbiol. Lett. 10, 395-397]. The quantity of DUSF is measured by the amount of [3H]DNA uptake by Neurospora cells at standard conditions. Its relative molecular mass was 230,000. It has an isoelectric point of pH 5.5. This protein consists of two identical subunits, relative molecular mass 110,000.     

Microspectrophotometric determination of the relative contents of nucleic acids in Pinus silvestris pollen

Based on inhibitor studies it has been concluded that in photoregulated processes of plant development, light induces differential gene expression. Using affinity chromatography of double labelled polysomal RNA on poly(U)-sepharose 4B, we were able to demonstrate different ³H/¹⁴C ratios for the bound poly(A) containing fraction (mRNA) when compared with the unbound fraction: when [³H]uridine was present in the light induced sample and [¹⁴C]uridine in the dark control, in the bound material the ³H/¹⁴C ratio was found to be higher than in the unbound fraction and vice versa. No such shift was observed, when both sample and control were kept in the dark. Our data are interpreted to provide evidence for photoinduced de novo synthesis of mRNA.Abbreviations CHI cycloheximide - CTAB N-acetyl-N,N,N-trimethylammoniumbromide - poly(A) poly-adenylic acid - Butyl-PBD 2-(4-t-Butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole