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.

Characterization of the expression products of recombinant human choriogonadotropin and subunits.

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.

Expression of human choriogonadotropin in monkey cells using a single simian virus 40 vector.

We have inserted the cDNAs coding for both polypeptide subunits, alpha and beta, of human choriogonadotropin (hCG) into a single simian virus 40 expression vector in such a way that they replace the viral VP2 and VP1 coding sequences, respectively. Monkey cells infected with this virus and the appropriate helper virus produce dimeric hCG. hCG produced in this system was shown to chromatograph identically to standard hCG preparations on gel filtration and to be biologically active in the mouse uterine weight assay.

Mammalian mutagenesis: future directions.

Future research in mammalian germ-line mutagenesis will benefit from the reciprocal relationship that can exist between the production of mutations and their analysis. Thus, mutagenesis experiments supply altered genes, and thereby tools for basic studies; while, in turn, information on gene structure and function helps in understanding mechanisms of mutagenesis. The nature of genetic alterations recovered in specific-locus tests is illustrated by an account of the analysis of 112 radiation-induced mutations involving the c locus on chromosome 7. Using phenotypic characterizations of various kinds, deficiency mapping with nearby markers, and complementation studies, the mutants could be classed into 13 groups, and 8 functional units could be identified in the c-locus region. Twelve different deficiencies overlapping at c range in length from less than 2 to 6-11 cM. Using the deficiencies, as well as a tandem duplication that involves the c region, and several T(X;7)'s in which c is inactivated in a mosaic fashion, it is possible to generate gene doses from 0 to 3, in steps of 0.5, not only for c but also for other genes included in various deficiencies. Cis and trans configurations can also be compared. This array of genetic materials is now being used for the isolation of DNA sequences from genetically-defined regions. Results from analyses of mutations can contribute answers to some of the pragmatic questions in germ-line mutagenesis and risk assessment. Areas in which contributions may be expected are: the relative roles of intracellular conditions (e.g., nature of chromatin, presence of repair enzymes) and secondary circumstances (e.g., selection) in determining the quantity and quality of transmitted mutations; the validity of quantitative extrapolations, such as projections to low doses and calculations of doubling dose; and the relation between measures of mutation rate and projections of phenotypic damage.

A cis-active regulatory gene in the mouse: direct demonstration of cis-active control of the rate of enzyme subunit synthesis.

Mouse mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] is a tetrameric protein. Two alleles of the structural gene (Mod-2) are known which code for electrophoretically distinct enzyme subunits: Mod-2a and Mod-2b. A regulatory gene (Mdr-1), closely linked to Mod-2 on chromosome 7, determines the rate of mitochondrial malic enzyme synthesis in brain. Two alleles of Mdr-1 are known: Mdr-1a (high activity) and Mdr-1b (low activitiy). By pulse-labeling with [35S]methionine, immune precipitation, and isoelectric focusing under dissociating conditions, we have measured the relative rates of synthesis of the two types of enzyme subunit in animals of genotypes Mdr-1a Mod-2a/Mdr-1a Mod-2b and Mdr-1S Mod-2a/Mdr-1b Mod-2b. The results show that in the former animals both types of subunit are made at an identical rate, whereas in the latter animals the Mod-2a gene product is synthesized at a rate 2.2 times that of the Mod-2b-coded subunit. Thus we have unambiguously demonstrated that Mdr-1 is cis-active in its control of the expression of the Mod-2 structural gene.

Regulation of mitochondrial malic enzyme synthesis in mouse brain.

In a previous study [Bernstine, E.G. (1979) J. Biol. Chem. 254, 83-87] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controlling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.

Enhanced expression of alkaline phosphatase in hybrids between neuroblastoma and embryonal carcinoma.

Three independent hybrid cell lines were isolated from the fusion of clonal lines of embryonal carcinoma and neuroblastoma. A series of subclones was subsequently derived from the original hybrid clones. In early hybrid generations all hybrid lines showed enhancement of alkaline phosphatase activity, expressing 2--8 times the activity of the teratoma parental line. The overexpression of APase appears to take place in the stationary phase of the growth cycle. Segregation for very high levels of APase activity was observed among subclones of one hybrid line. Specific activities of the segregants ranged from 0.1 to 133. Results of heat denaturation studies are consistent with the hypothesis that it is the embryonal carcinoma APase that is being expressed in the hybrids.