Successful induction of immune tolerance to enzyme replacement therapy in canine mucopolysaccharidosis I.

Immune responses can interfere with the effective use of therapeutic proteins to treat genetic deficiencies and have been challenging to manage. To address this problem, we adapted and studied methods of immune tolerance used in canine organ transplantation research to soluble protein therapeutics. A tolerization regimen was developed that prevents a strong antibody response to the enzyme alpha-l-iduronidase during enzyme replacement therapy of a canine model of the lysosomal storage disorder mucopolysaccharidosis I. The tolerizing regimen consists of a limited 60-day course of cyclosporin A and azathioprine combined with weekly i.v. infusions of low-dose recombinant human alpha-l-iduronidase. The canines tolerized with this regimen maintain a reduced immune response for up to 6 months despite weekly therapeutic doses of enzyme in the absence of immunosuppressive drugs. Successful tolerization depended on high plasma levels of cyclosporin A combined with azathioprine. In addition, the induction of tolerance may require mannose 6-phosphate receptor-mediated uptake because alpha-l-iduronidase and alpha-glucosidase induced tolerance with the drug regimen whereas ovalbumin and dephosphorylated alpha-l-iduronidase did not. This tolerization method should be applicable to the treatment of other lysosomal storage disorders and provides a strategy to consider for other nontoleragenic therapeutic proteins and autoimmune diseases.

REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase.

We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.

Increased mdr gene expression and decreased drug accumulation in multidrug-resistant human melanoma cells.

Multidrug-resistant clones of a drug-sensitive human malignant melanoma cell line were isolated by single-step selection in culture medium containing either vincristine (4.5 ng/ml or 7.5 ng/ml), vinblastine (3 ng/ml), or colchicine (8 ng/ml). This protocol yielded primary colonies showing relatively low (4- to 24-fold) levels of drug resistance. These clones exhibit the classical multidrug resistance (MDR) phenotype, being cross-resistant to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D. The appearance of an MDR phenotype in these cells was linked to a decreased accumulation and increased efflux of the drug [3H]vinblastine when compared to the drug-sensitive melanoma cell line. This increased drug efflux was dependent on the presence of cellular ATP and could be reduced by treatment of the cells with rotenone and deoxyglucose. A partial human mdr complementary DNA clone was used to monitor the degree of amplification and the level of transcription of this gene in the cloned lines. All 5 MDR sublines expressed increased levels of the specific 4.5-kilobase mdr mRNA, but did not show mdr gene amplification. Our results indicate that relatively low levels of drug resistance, similar to those observed clinically and in experimental xenografts, can be achieved by single-step drug selection and result from increased expression of at least one member of the mdr gene family.

Expression of active human uterine tissue plasminogen activator in yeast.

Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.

Function and autoregulation of yeast copperthionein.

The CUP1 gene of yeast encodes a small, metallothionein-like protein that binds to and is inducible by copper. A gene replacement experiment shows that this protein protects cells against copper poisoning but is dispensable for normal cellular growth and development throughout the yeast life cycle. The transcription of CUP1 is negatively autoregulated. This feedback mechanism, which is mediated through upstream control sequences, may play an important role in heavy metal homeostasis.

Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE.

The umr7-1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7-1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and alpha cells and has no apparent effect on a mating functions. However, alpha umr7 meiotic segregants fail to express several alpha-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone alpha-factor and response to the hormone a-factor). In addition, alpha umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar(+)) that prevents diffusion of alpha-factor and an increased mating frequency with alpha strains. The most striking property of alpha umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with alpha-factor. Some a/alpha-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/alpha umr7/umr7 diploids have medial budding like a/a, alpha/alpha and haploid strains. Moreover, a/alpha umr7/umr7 diploids have lost the ability to sporulate and are Bar(+) like a or a/a strains. Revertant studies indicate that umr7-1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.

Genetic and physiological factors affecting repair and mutagenesis in yeast.

Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various "pathways" of repair are described, particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as "cell age," DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

Effects of spermine on the detection of induced forward mutation at the Can1 locus in yeast: evidence for selection against canavanine-resistant mutants.

The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance (CAN1 leads to can1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, a significant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract--peptone--dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures "recovering" from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolate revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.

Induced mutagenesis in Ustilago maydis. I. Isolation and characterization of a radiation-revertible allele of the structural gene for nitrate reductase.

A UV-revertible mutant of the nar1 structural gene for nitrate reductase was isolated in wild-type (nar+ nir+) Ustilago maydis. It proved to be vigorously revertible by gamma rays as well. Genetic analysis revealed that the strain carried a single, nonleaky, recessive allele (nar1-m) with an unusually high spontaneous reversion rate (approximately 3 X 10(-5)/div.). Reliable reversion frequencies were determined with a special agar medium that reduced the normally high level of residual growth observed on nitrate minimal agar. Radiation-induced reversion frequencies in the homozygous diploid were approximately twice those in the haploid. Following crosses to wild type, two revertants (one spontaneous and one UV-induced) were found to map at nar1. Although the molecular basis of nar1-m reversion is not known, available data suggest that some form of point mutation is involved.

Induced mutagenesis in Ustilago maydis. II. An in vivo biochemical assay.

UV gamma radiation-induced reversion to nar+ in a nar1-m nir1-1 strain of Ustilago maydis was found to occur under nongrowth conditions by performing the in vivo assay for functional nitrate reductase described by Resnick and Holliday (1971) who previously demonstrated that nonviable cells may still synthesize normal or near-normal levels of activity. Reversion frequencies of a signle gamma-irradiated culture were estimated in two cell populations by different methods: (A) among surviving clones after plating, and (B) among all cells (viable and nonviable) in suspension in the absence of postirradiation cell division. At gamma doses (300, 500 krad) corresponding to considerable cell killing (35%, 2% survival), reversion frequency by either method was the same. This supports the conclusion that mutation induction by gamma rays and its expression occur in nonviable cells with the same frequency as among survivors. If an error-prone repair system is assumed to be responsible for the observed gamma revertibility, then it is argued that this process is constituitive rather than inducible and that it is recombination-independent.