Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein α subunits. The Ric-8A isoform chaperones Gαi/o, Gα12/13, and Gαq/11 subunits, while Ric-8B acts on Gαs/olf subunits. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ric-8B in complex with Gαs and Gαolf, revealing isoform differences in the relative positioning and contacts between the C-terminal α5 helix of Gα within the concave pocket formed by Ric-8 α-helical repeat elements. Despite the overall architectural similarity with our earlier structures of Ric-8A complexed to Gαq and Gαi1, Ric-8B distinctly accommodates an extended loop found only in Gαs/olf proteins. The structures, along with results from Ric-8 protein thermal stability assays and cell-based Gαolf folding assays, support a requirement for the Gα C-terminal region for binding specificity, and highlight that multiple structural elements impart specificity for Ric-8/G protein binding.

We used a shuttle vector based on the Epstein‐Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40°C for various times to induce up to 20 apurinic (AP) sites per 9.7‐kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid‐encoded herpes simplex virus type 1‐thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10‐fold to 13‐fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46–55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100–150 bp) and characterized by short (1–5 bp) regions of sequence homology at the end‐points. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP‐site‐induced template dislocation during DNA synthesis. A broad spectrum of base‐substitution mutations, which accounted for 19–36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43–55%) > A°C (18–21%) > T (9–14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.© 1992 Wiley‐Liss, Inc.

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt+/−→aprt−/−) was 1.3 × 10−5/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.

Mutational activation of cellular proto‐oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the on P‐tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N ‐ethyl‐ N ‐nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein‐Barr virus (EBV) and is stably maintained as a plasmid in EBV‐transformed human lymphoblastoid cells. Populations of plasmid‐bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7–8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV‐tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC→Ã transitions and 17% were AT→GC transitions. In addition, the number of AT→TA (20%) and AT→CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O 2 ‐ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT→TA transversion mutations in the activation of cellular proto‐oncogenes by ENU.

We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens. The vector is able to replicate as a plasmid in Escherichia coli and in Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies. After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance. Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5). Treatment of plasmid-bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene. The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase.