A Comprehensive Analysis of the Importance of Translation Initiation Factors for Haloferax volcanii Applying Deletion and Conditional Depletion Mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.     

Selective Growth Inhibition of Sphaerotilus natans and Beggiatoa sp. by Nucleosides

9-beta-d-Arabinofuranosyladenine (ara-A) has been found to specifically inhibit the growth of Sphaerotilus natans and Beggiatoa sp. at a low concentration (0.78 mug/ml). The nucleoside had no antimicrobial activity against various microorganisms other than Candida albicans at 1,000 mug/ml. 3'-Deoxyadenosine, 2'-deoxyadenosine, formycin, and some derivatives of ara-A also showed inhibitory activity against Sphaerotilus natans. The growth of Beggiatoa sp. was also inhibited by 9-beta-arabinofuranosylhypoxanthine, 3'-deoxyadenosine, 2'-deoxyadenosine, formycin, toyocamycin, tubercidin, and some derivatives of ara-A. ara-A was quite stable in water and had no harmful effect on fish at 200 mug/ml. The possible uses of ara-A and some nucleosides in controlling the proliferation of S. natans and Beggiatoa sp. in the environment are discussed.     

Isolation and Genetic Analysis of Extragenic Suppressors of the Hyper-Deletion Phenotype of the Saccharomyces Cerevisiae Hpr1δ Mutation

The HPR1 gene of Saccharomyces cerevisae is involved in maintaining low levels of deletions between DNA repeats. To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S. cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1δ mutation. The mutations defined five different genes called HRS for hyper-recombination suppression. They suppress the hyper-deletion phenotype of hpr1δ strains for three direct repeat systems tested. The mutations eliminated the hyper-deletion phenotype of hpr1δ strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1). None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions. Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1δ mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1δ and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1δ strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated. We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.     

2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Genetic analysis of the susceptibility of mouse cytomegalovirus to acyclovir.

Eight independently derived mouse cytomegalovirus (MCMV) mutants resistant to acyclovir (ACV) were obtained by the sequential plating of wild-type virus in increasing concentrations of ACV. Results of complementation studies among these eight mutants suggest that all had mutations within the same or closely associated genes. A ninth MCMV mutant resistant to phosphonoacetate (PAA) derived by plating wild-type virus in the presence of 100 micrograms of PAA per ml displayed coresistance to ACV and was unable to complement any of the ACV-derived mutants. Recombination experiments among all combinations of the nine MCMV mutants were performed and supported the complementation data in that no recombination could be detected. Seven of the eight ACV-resistant mutants demonstrated cross-resistance to PAA and hypersensitivity to aphidicolin. The one mutant not coresistant to PAA was more susceptible to PAA than was the parent virus. Only a few mutants demonstrated coresistance when the mutants were tested against 9-beta-D-arabinofuranosyladenine (ara-A). The ACV mutant that demonstrated increased susceptibility to PAA was 30-fold more susceptible to ara-A but remained unchanged in susceptibility to aphidicolin. Two of the parent-mutant combinations were selected for DNA synthesis analysis in the presence of ACV (5 microM). A significant decrease in DNA synthesis was demonstrated for both parent viruses, and there was little effect on mutant virus DNA synthesis at the same drug concentration. These results suggest that susceptibility of MCMV to ACV is confined to a product of a single gene and that a mutation of this gene can lead to an altered phenotype when compared with parent virus in susceptibility of DNA synthesis to PAA, ara-A, and aphidicolin, drugs that are known to inhibit DNA polymerase activity.     

Mutator Phenotype Induced by Aberrant Replication

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase δ (polδ) and mutants of Polα, two Polδ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2⁺, which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Δ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Δ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Δ. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.     

Regioselective halogenation of some 2,3-anhydro pyrimidine nucleosides with dilithium tetrahalocuprates.

The reaction of 1-(2,3-anhydro-5-O-trityl-beta-D-lyxofuranosyl)-2-O-methyluracil (1a) and its thymine analogue (1b) with dilithium tetrahalocuprate (Li2CuX4) revealed excellent to perfect regioselectivity, yielding 2,2'-anhydro-3'-halonucleosides (2a-d), while the same reactions with 2,3-anhydro uracil and thymine nucleosides (4a,b) gave arabinosyl (5a-d) and xylosyl halohydrins (6a-d) with the respective product ratio of 7:3 to 8:2. compounds 5 and 6 were isolated as the 2-O-(7) and 3- O-mesyl derivatives (8).     

A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a; however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.     

Synthesis and reactivities of 2,2'-anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl- beta-D-arabinofuranosyl)thymine.

2,2'-Anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl-beta-D-arabinofuranosyl) thymine (2) was synthesized from 2',3'-didehydro-3'-deoxythymidine (DHT). Compound 2 was readily converted into the 2',3'-anhydrolyxofuranosyl derivatives 4-6. Treatment of 4a with some nucleophiles (N3-, OMe-, Cl-) gave the corresponding 3'-substituted arabinosyl nucleosides (7a,c,e) together with the minor xylosyl isomers (8a,c,d). 7a,c,e were deprotected to 7b,d,f, respectively.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

Reactions of Some 2,3-Anhydro Pyrimidine Nucleosides with Dilithium Tetrahalocuprates

Abstract

The reaction of 1-(2,3-anhydro-5-0-trityl-β-D-lyxofuranosyl)-2-0-methyluracil (2a) and its thymine analogue (2b) with dilithium tetrahalocuprates (Li₂CuX₄) revealed an excellent to perfect regioselectivity, yielding 2,2′-anhydro-3′-halonucleosides (3a-d), while the same reactions with 2,3-anhdro uracil and thymine nucleosides (5a,b) gave arabinosyl (6a-d) and xylosyl halohydrins (7a-d) with respective product ratios of 7:3 to 8:2 which were estimated after mesylation to 8a-d and 9a-d.     

The Mitochondrial Genome Integrity Gene, Mgi1, of Kluyveromyces Lactis Encodes the β-Subunit of F(1)-Atpase

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F(1)-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F(1) complex is needed for the ``gain-of-function' phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F(1)-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm(5)s(2)U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2)U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.     

Analysis of mutational effects at the HPRT locus in human G(0) phase lymphocytes irradiated in vitro with gamma rays

The mutational effects of ionising radiation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were studied in human peripheral blood G(0) phase lymphocytes irradiated in vitro with gamma rays. The presence of radiation induced mutants was assessed by selecting the HPRT mutants every week on the basis of 6-thioguanine resistance up to 1 month after irradiation. A dose-related increase of 14.25x10(-6) mutants/Gy was measured after an expression time of 7 days. After 2 weeks from culture starting the fraction of clonable cells in irradiated and control cell populations decreased, limiting the measurements of mutant frequency. The mutational spectrum of the HPRT gene was determined by PCR analyses in a total of 99 mutant clones derived from irradiated lymphocytes. The independent origin of mutant clones carrying the same mutation was assessed by analysing the TCR gamma gene rearrangements. The results showed a dose-related increase of deletion mutants up to 3Gy, whereas point mutation frequency increased only up to 2Gy. Two preferentially deleted regions were identified; one involving the HPRT exon 3, and another one the 3'-terminal and the 3'-flanking region of the gene. One complex mutation involving a non-contiguous deletion of exons 2-5 and 7/8 was observed among the mutants isolated after 3Gy irradiation.     

Effect of 9-beta-D-arabinofuranosyladenine on polymer synthesis in a polyauxotrophic strain of Escherichia coli

Leung, Hazel Barner (University of Pennsylvania School of Medicine, Philadelphia), Alice McGovern Doering, and Seymour S. Cohen. Effect of 9-beta-d-arabinofuranosyladenine on polymer synthesis in a polyauxotrophic strain of Escherichia coli. J. Bacteriol. 92:558-564. 1966.-Adenine-requiring mutants have been obtained from Escherichia coli strain 15 TAU, which also needs thymine, arginine, and uracil for growth. Some of these are killed by 9-beta-d-arabinofuranosyladenine (ara-A) in the absence of exogenous adenine; a particular mutant of this type, designated TAUAd, has been used in our studies. The lethality of ara-A, d-arabinosylhypoxanthine, and the 1-n-oxide of ara-A has been compared; ara-A is equally toxic in the presence or absence of thymine. Although the absence of uracil reduces ara-A toxicity, the lack of arginine almost eliminates lethality. It was found that ara-A completely inhibits deoxyribonucleic acid synthesis without markedly affecting ribonucleic acid (RNA) synthesis. Some inhibition of protein synthesis can be detected. However, the interpretation of these results is complicated because (i) exogenous adenine must be excluded, (ii) endogenous adenine is made available from RNA turnover, and (iii) ara-A is being rapidly converted to only slightly less toxic arabinosylhypoxanthine by the adenosine deaminase of E. coli. A suitable inhibitor for the bacterial deaminase has not yet been found.     

Sesquiterpene polyol esters from Celastrus rosthornianus

Two new sesquiterpene polyol esters with β-dihydroagarofuran skeleton were isolated from the root bark of Celastrus rosthornianus. Their structures were elucidated, mainly on the basis of spectral analyses, as 1 β-acetoxy-8β,9-dibenzoyloxy-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran and 1β-acetoxy-9-benzoyloxy-8β-(β-furanocarbonyloxy)-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran. The complete assignments of ¹³C NMR chemical shifts for both compounds on the basis of ¹H-¹³C chemical-shift correlation spectrum were also carried out.     

Synthesis of part of the antigenic repeating-unit of streptococcus pneumoniae type II

Condensation of methyl 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranoside with 2,3,4,6-tetra-O-benzyl-α- -glucopyranosyl chloride gave a mixture of methyl O-[2,3,4,6-tetra-O-benzyl-α- (4) and -β- -glucopyranosyl]-(1→2)-O-[(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-(1→3)]-4-O-acetyl-α- -rhamnopyranoside (9) in 43:7 proportion in 63% yield. After chromatographic separation, removal of the benzyl and acetyl groups gave methyl O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside and the β anomer. Removal of benzyl groups of 4 was followed by tritylation, acetylation, and detritylation of the α- -glucopyranosyl group, and finally condensation with benzyl (2,3,4-tri-O-benzyl- -glucopyranosyl chloride)uronate gave a mixture of two tetrasaccharides (15 and 16), containing the α- and β- -glucopyranosyluronic acid groups in the ratio 81:19, and an overall yield of 71%. After chromatographic separation, alkaline hydrolysis and hydrogenation of 15 gave methyl O-α- -glucopyranosyluronic acid-(1→6)-O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside. The β- anomer was obtained by similar treatment of 16. 6-O-α- -glucopyranosyluronic acid-α,β- -glucopyranose was synthesized as a model compound.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.