Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N- acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM- resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.     

Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells.

We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11. The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency. A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2. The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa. This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene. The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein. Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin. Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.     

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin

Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells. A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N-glycan is involved in the binding. Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin. In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan. Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein

Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C.     

A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS.     

Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5-(2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

Physiological and morphological effects of overproduction of membrane-bound ATP synthase in Escherichia coli K-12.

Effects of increased biosynthesis of the membrane-bound ATP synthase of Escherichia coli K-12 were analysed at the physiological and morphological level. Overproduction of the enzyme complex was achieved by molecular cloning of the structural genes into plasmid pBR322. A series of plasmids resulting in 2-fold, 4- to 5-fold and 10- to 12-fold overproduction, respectively, was constructed. The ATP synthase was calculated to represent 3%, 6-7% and 18-23%, respectively, of total protein in cells with these plasmids. In wild-type cells ATP synthase represents 1.5-2% of total protein equivalent to approximately 3000 enzyme complexes per average cell. While 2- or 4- to 5-fold wild-type levels of the ATP synthase had only minor effects it was found that 10- to 12-fold overproduction resulted in pronounced inhibition of cell division and growth and in formation of membrane cisterns and vesicles within the cells. Inclusion bodies, probably representing deposits of excess ATP synthase, were also observed in these cells.     

Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats

The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D₂ synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G₁ to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21^Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes. apoptosis; atherosclerosis; insulin resistance     

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.     

Amplification of the UMP synthase gene and enzyme overproduction in pyrazofurin-resistant rat hepatoma cells. Molecular cloning of a cDNA for UMP synthase

The levels of UMP synthase protein and mRNA are increased in rat hepatoma cells that have acquired resistance to pyrazofurin, a potent inhibitor of pyrimidine biosynthesis. A cDNA plasmid library was prepared from partially purified poly(A)+ mRNA isolated from the resistant cell line. Recombinant plasmids with inserts complementary to UMP synthase mRNA were selected by differential hybridization with cDNA prepared from wild type and resistant cell mRNA and analysis of hybrid-selected mRNA by in vitro translation reactions. One plasmid, pUMPS-2, contains a 850-base pair insert and was used to analyze UMP synthase gene sequences in the wild type and resistant cell lines. Blot hybridization of restricted genomic DNA demonstrated amplification of the UMP synthase gene in the resistant cells. The number of UMP synthase genes is increased 15-fold as determined by a modified dot hybridization procedure. Previous studies have shown that the resistant cells have a 16-fold increase in UMP synthase mRNA but a 40-fold increase in synthase activity (Suttle, D.P. (1983) J. Biol. Chem. 258, 7707-7713). To further investigate this discrepancy between the amount of increase in DNA and mRNA versus the increase in enzyme activity, we have determined the relative rate of synthesis and degradation of UMP synthase. The rate of synthesis was 13-fold faster in the resistant cells. The degradation rate was not significantly different between the two cell lines. These data indicate that gene amplification is the major factor contributing to the enzyme overproduction in the pyrazofurin-resistant cells.     

Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin

The biochemical basis for the inhibition of fatty acid biosynthesis in Escherichia coli by the antibiotic thiolactomycin was investigated. A biochemical assay was developed to measure acetoacetyl-acyl carrier protein (ACP) synthase activity, a recently discovered third condensing enzyme from E. coli (Jackowski, S., and Rock, C.O. (1987) J. Biol. Chem. 262, 7927-7931). In contrast to the other two condensing enzymes in E. coli, acetoacetyl-ACP synthase (synthase III) condensed malonyl-ACP with acetyl-CoA, rather than with acetyl-ACP. The concentration dependence of thiolactomycin inhibition of fatty acid biosynthesis in vivo was the same as the inhibition of acetoacetyl-ACP synthase activity in vitro indicating that the two phenomena were related. A thiolactomycin-resistant mutant (strain CDM5) was isolated. The specific activity of acetoacetyl-ACP synthase in extracts from this mutant was 10-fold lower than in extracts from its thiolactomycin-sensitive parent resulting in a marked defect in the ability of strain CDM5 to incorporate acetyl-CoA into fatty acids in vitro. The residual acetoacetyl-ACP synthase activity in the resistant strain was refractory to thiolactomycin inhibition. In addition, acetyl-CoA:ACP transacylase activity in strain CDM5 was resistant to inactivation by thiolactomycin suggesting that the acetoacetyl-ACP synthase also catalyzes this transacylation reaction. These data point to acetoacetyl-ACP synthase as a target for thiolactomycin inhibition of bacterial fatty acid biosynthesis.     

Tunicamycin-resistant mutations in mouse FM3A cells

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 μg/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1–1.2 μg tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10⁻⁴ to 10⁻⁵ in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 μg N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 × 10⁻⁶ per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D₁₀ value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [³H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.     

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.