Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild- type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.     

Isolation and characterization of an extragenic suppressor of the low-density lipoprotein receptor-deficient phenotype of a Chinese hamster ovary cell mutant.

ldlC cells are low-density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants which express pleiotropic defects in Golgi-associated glycosylation reactions. The dramatically reduced stability of the abnormally glycosylated LDL receptors in ldlC cells was shown to be due, in part, to rapid proteolysis and release of a large extracellular fragment of the receptor into the medium. A set of spontaneously arising LDL receptor-positive revertants of ldlC cells has been isolated. One of these, RevC-13, exhibits the glycosylation defects characteristic of the original ldlC mutant, suggesting that restoration of receptor activity was due to extragenic suppression. This suppression was due to a dramatic increase in the rate of LDL receptor synthesis rather than to an increase in the stability of the abnormally glycosylated receptors. Increased receptor synthesis was not due to receptor gene amplification. The increased LDL receptor activity was subject to normal sterol regulation. Analysis of the RevC-13 extragenic suppressor activity in a series of hybrid cells showed that RevC-13 suppression was a codominant trait that acted in cis to the LDL receptor structural gene (ldlA). Thus, the extragenic suppression in RevC-13 cells has defined a genetic element which is either part of or linked to the LDL receptor structural gene and which can control LDL receptor expression.     

A new class mutation of low density lipoprotein receptor with altered carbohydrate chains

In a monensin-resistant mutant (Monr-31) of Chinese hamster ovary cells, the O-linked sugar chains of the low density lipoprotein (LDL) receptor are altered, suggesting a mutation at a Golgi apparatus gene. In a compactin-resistant mutant (MF-2) of Chinese hamster V79 cells, the mature LDL receptor is apparently 5000 daltons smaller; the difference is due to altered glycosylation of O-linked sugar chains. Hybrids between MF-2 and Monr-31 still produced LDL receptor molecules with aberrant sugar chains; thus both mutants are in the same complementation group. Krieger and his colleagues (Krieger, M., Kingsley, D., Sege, R., Hobbie, L., and Kozarsky, K. (1985) Trends. Biochem. Sci. 10, 447-452) have classified Chinese hamster ovary cell mutants with altered LDL receptor structure into four groups: ldlA, ldlB, ldlC, and ldlD. Cell-cell hybrids between their ldl mutants and Monr-31 produced wild type mature LDL receptors with normal molecular sizes, suggesting that these compactin- and monensin-resistant mutants define a new class of LDL receptor mutant. Since both of our mutants are defective in internalization of LDL, we assign them as int mutants. This may imply a further etiology for hypercholesterolemia, and cases can now be examined for such a class.     

Scavenger receptor,Class B,Type I provides an alternative means for β-VLDL uptake independent of the LDL receptor in tissue culture

Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate β-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from β-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of β-VLDL was ∼ 3 times higher after SR-BI over-expression, which was competed by β-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated β-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled β-VLDL. β-VLDL was degraded at a rate of 6 ng β-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, β-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in β-VLDL uptake. β-VLDL degradation was similar to ldlA7-SRBI cells. When β-VLDL uptake was followed using fluorescence microscopy, β-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.     

Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid- linked carbohydrate chains

Biochemical, immunological, and genetic techniques were used to investigate the genetic defects in three types of low density lipoprotein (LDL) receptor-deficient hamster cells. The previously isolated ldlB, ldlC, and ldlD mutants all synthesized essentially normal amounts of a 125,000-D precursor form of the LDL receptor, but were unable to process this receptor to the mature form of 155,000 D. Instead, these mutants produced abnormally small, heterogeneous receptors that reached the cell surface but were rapidly degraded thereafter. The abnormal sizes of the LDL receptors in these cells were due to defective processing of the LDL receptor's N- and O-linked carbohydrate chains. Processing defects in these cells appeared to be general since the ldlB, ldlC, and ldlD mutants also showed defective glycosylation of a viral glycoprotein, alterations in glycolipid synthesis, and changes in resistance to several toxic lectins. Preliminary structural studies suggested that these cells had defects in multiple stages of the Golgi-associated processing reactions responsible for synthesis of glycolipids and in the N-linked and O-linked carbohydrate chains of glycoproteins. Comparisons between the ldl mutants and a large number of previously isolated CHO glycosylation defective mutants showed that the genetic defects in ldlB, ldlC, and ldlD cells were unique and that only very specific types of carbohydrate alteration could dramatically affect LDL receptor function.     

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.     

Complementation of mutations in the LDL pathway of receptor-mediated endocytosis by cocultivation of LDL receptor-defective hamster cell mutants

We have previously isolated Chinese hamster ovary (CHO) cell mutants that do not express low density lipoprotein (LDL) receptors. When one mutant clone was cocultivated with other receptor-defective clones, it was induced to express receptors that could mediate normal endocytosis. These LDL receptor-defective clones defined two classes of mutations: cbc (complemented by cocultivation) and icc (inducer cells in cocultivation). The induction and short-term (18 hr) stability of LDL receptors in cbc cells did not require protein synthesis by icc cells. Receptor activity could not be induced by DMSO, 5-azacytidine, phosphatidylcholine liposomes, dibutyryl cAMP, compactin, soybean trypsin inhibitor, low temperature (30°C), or conditioned medium, but could be induced by cocultivation with parental CHO cells and normal and LDL receptor-negative human fibroblasts. Complementation by cocultivation only occurred when the cbc and inducing cells were in close proximity, suggesting that an unstable diffusible factor or intimate cell-to-cell association was required for complementation.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Chinese hamster ovary cell mutant with defective down-regulation of low density lipoprotein receptors

A monensin-resistant clone (Monr-31) shows a related series of differences from its parental Chinese hamster ovary (CHO) cell line in the cellular response to several ligands. The uptake and metabolism of low density lipoprotein (LDL) in the mutant cells are defective. Accumulation of fluorescent-labeled LDL as well as internalization and degradation of 125I-LDL are greatly reduced in Monr-31 cells. The receptor number for LDL on the cell surface of Monr-31 is about one-third that for CHO cells, but affinity constants for both cell lines are similar. Electrophoretic analysis shows a slightly reduced molecular weight of LDL receptor in Monr-31 cells in comparison to that in CHO cells. The internalization index (internalization plus degradation per binding) of LDL of the mutant is about one-half that of CHO cells, suggesting a failure of internalization of LDL as well as LDL binding. Hybrids (hyb-1, -2, and -3) between CHO and Monr-31 cells show LDL binding and LDL internalization activities comparable to that of CHO cells, suggesting that the altered LDL response in Monr-31 cells is recessive. Addition of exogenous LDL to culture medium down-regulates the LDL receptor activity of CHO, hyb-2, and hyb-3 cells, whereas no such down-regulation is seen in Monr-31 cells. Probably as a result of the failure of down-regulation, the prominent inhibition of sterol synthesis from acetate and 3-hydroxy-3-methylglutaryl-coenzyme A reductase observed in CHO cells is scarcely detectable in Monr-31 cells. As a correlated result, sterol synthesis from acetate is 6-fold higher in the mutant. The failure of down-regulation of LDL receptors in Monr-31 cells is discussed in relation to the altered binding and internalization of LDL.     

The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides

The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.     

Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.

The scavenger receptor-BI (SR-BI) delivers sterols from circulating lipoproteins to tissues, but the relative potency of individual lipoproteins and the transported cholesterol has not been studied in detail. In this study, we used Chinese hamster ovary cells that express recombinant mouse SR-BI but have no functional low density lipoprotein (LDL) receptors (ldlA7-SRBI cells) to compare the fate of lipids transferred from high or low density lipoproteins to cells by SR-BI. HDL and LDL were equally effective in mediating the transfer of [(3)H]cholesterol to cells. Only 5% of the free cholesterol transferred to cells was esterified, in direct contrast to the findings in the cells that express LDL receptors in which 50% of the transported cholesterol was esterified. Almost all the free cholesterol transferred from lipoproteins to cells was rapidly excreted when the ldlA7-SRBI cells were switched to media containing unlabeled lipoproteins. SR-BI expression was associated with an increase in selective cholesteryl ester uptake from both lipoproteins, but HDL was a more effective donor. HDL and LDL were equally effective in delivering cholesterol to the intracellular regulatory pool via SR-BI. These data indicate that SR-BI is able to exchange cholesterol rapidly between lipoproteins and cell membranes and can mediate the uptake of cholesteryl esters from both classes of lipoproteins.     

Transforming growth factor-beta (TGF-beta) receptor proteoglycan. Cell surface expression and ligand binding in the absence of glycosaminoglycan chains

The type III transforming growth factor-beta (TGF-beta) receptor is a cell surface chondroitin/heparan sulfate proteoglycan that binds various forms of TGF-beta with high affinity and specificity. Here, we have used a genetic approach to determine the requirement for glycosaminoglycan (GAG) chains for normal TGF-beta receptor expression and the role that the receptor proteoglycan core and GAG chains play in TGF-beta binding. Chinese hamster ovary (CHO) cells defective in GAG synthesis express on their surface 110-130-kDa type III receptor proteoglycan cores that can bind normal levels of TGF-beta compared to wild type CHO cells. The affinity of the receptor core for TGF-beta 1 and TGF-beta 2 in CHO cell mutants is similar to that of the TGF-beta receptor proteoglycan forms present in wild type CHO cells or in CHO cell mutants that have been allowed to bypass their metabolic defect and express the wild type proteoglycan phenotype. The binding properties of TGF-beta receptor types I and II in CHO cells and the growth-inhibitory response of CHO cell mutants to TGF-beta are not impaired by the absence of GAG chains in the type III receptor. These results show that the GAG chains are dispensable for type III receptor expression on the cell surface, binding of TGF-beta to the receptor core, and growth inhibitory response of the cells to TGF-beta. The evidence also suggests that the type III receptor may act as a multifunctional proteoglycan able to bind TGF-beta via the receptor core while performing another as yet unidentified function(s) via the GAG chains.     

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.     

The nature of ultraviolet light-induced mutations at the heterozygous aprt locus in Chinese hamster ovary cells

A system for studying mutational specificity at a heterozygous locus in Chinese hamster ovary (CHO) cells is described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase (aprt) alleles. We have demonstrated the usefulness of this approach through the characterization of a collection of aprt- mutants with respect to the role played by loss of heterozygosity events in ultraviolet light (UV) induced mutagenesis. A similar strategy has also been applied to speculate on the identity of the premutational lesion responsible for a UV-induced mutational hotspot at the aprt locus.     

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.     

Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: mutation at the adenosine kinase locus in Chinese hamster cells.

The pyrrolopyrimidine nucleosides toyocamycin and tubercidin show several unique features of growth inhibition in Chinese hamster ovary (CHO) cells. Stable mutants which are more than 600-fold resistant to these drugs are obtained in CHO cells at a strikingly high frequency of approximately 10(-3), in the absence of mutagenesis. The mutants resistant to toyocamycin (Toyr) and tubercidin (Tubr) exhibit similar cross-resistance patterns to the two selective drugs as well as to adenosine and 6-methyl mercaptopurine riboside, indicating that the same lesion is probably involved in all cases. The mutants examined were found to be deficient in the enzyme adenosine kinase (AK), indicating that the phosphorylation of these analogs is an essential first step in their toxic action. The above mutants (AK-) behaved recessively in cell hybrids, and segregation studies indicate that the AK locus is not linked to the X chromosome. The frequencies of similar Toyr mutants in other Chinese hamster lines, e.g., V79, CHW, M3-1, GM7, and CHO-K1, varied from similar to more than three logs less than that observed for CHO cells, indicating that various cell lines probably differ in the number of functional gene copies for this locus.     

LDLC encodes a brefeldin A-sensitive,peripheral Golgi protein required for normal Golgi function

Two genetically distinct classes of low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants, ldlB and ldlC, exhibit nearly identical pleiotropic defects in multiple medial and trans Golgi-associated processes (Kingsley, D., K. F. Kozarsky, M. Segal, and M. Krieger. 1986. J. Cell Biol. 102:1576-1585). In these mutants, the synthesis of virtually all N- and O-linked glycoproteins and of the major lipid-linked oligosaccharides is abnormal. The abnormal glycosylation of LDL receptors in ldlB and ldlC cells results in their dramatically reduced stability and thus very low LDL receptor activity. We have cloned and sequenced a human cDNA (LDLC) which corrects the mutant phenotypes of ldlC, but not ldlB, cells. Unlike wild-type CHO or ldlB cells, ldlC cells had virtually no detectable endogenous LDLC mRNA, indicating that LDLC is likely to be the normal human homologue of the defective gene in ldlC cells. The predicted sequence of the human LDLC protein (ldlCp, approximately 83 kD) is not similar to that of any known proteins, and contains no major common structural motifs such as transmembrane domains or an ER translocation signal sequence. We have also determined the sequence of the Caenorhabditis elegans ldlCp by cDNA cloning and sequencing. Its similarity to that of human ldlCp suggests that ldlCp mediates a well- conserved cellular function. Immunofluorescence studies with anti-ldlCp antibodies in mammalian cells established that ldlCp is a peripheral Golgi protein whose association with the Golgi is brefeldin A sensitive. In ldlB cells, ldlCp was expressed at normal levels; however, it was not associated with the Golgi. Thus, a combination of somatic cell and molecular genetics has identified a previously unrecognized protein, ldlCp, which is required for multiple Golgi functions and whose peripheral association with the Golgi is both LDLB dependent and brefeldin A sensitive.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.