Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Isolation and characterization of context mutations affecting the suppressibility of nonsense mutations

Summary Secondary mutations which increase the efficiency of suppression of nonsense mutations in the rHB cistron of bacteriophage T4 have been isolated. These secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. In context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. The context mutations examined can act on the UAA (ochre) nonsense allele as well as on the UAG (amber) nonsense allele at a given site. These context mutations affect all suppression mechanisms analyzed (genetic suppressors. 5-fluorouracil suppression and spontaneous suppression).We suggest that context mutations affect information which is significant to the termination of polypeptide chains. According to our view, context mutations change the immediate neighborhood of nonsense mutations and so reduce the degree of resemblance to the sequences normally used for the termination of translation.     

The role of sialic acid and galactose residues in determining the survival of human plasma alpha-antitrypsin in the blood circulation.

Human plasma α₁-antitrypsin (α₁-AT) is a glycoprotein known to contain terminal sialic acids (N-acetylneuraminic acids) in the carbohydrate units. These residues were converted to a radioactive seven-carbon analog (NANA-7) by sequential periodate oxidiation and tritiated borohydride reduction. Modified α₁-AT prepartions, namely, (a) periodate oxidized α₁-AT, (b) asialo α₁-AT (neuraminidase-treated α₁-AT), (c) (NANA 7)-α₁-AT (periodate-oxidized, -tritiated, borohydride-reduced α₁-AT), (d) (NANA-7)-α₁ AT (partially desialylated by neuraminidase), and (e) partially desialylated (NANA 7)-α₁-AT oxidized with galactose oxidase, all retained the following properties attributable to native α₁-AT: trypsin-inhibitory and chymotrypsin-inhibitory activities, immunological reactivity to antibody against native α₁-AT, and the ability to bind to concanavalin A-Sepharose 4-B columns. After intravenous injection of intact (NANA-7)-α₁-AT into rats, the labeled material had a circulating half-life of 18 h. When (NANA-7)-α₁-AT was partially desialylated (four residues of NANA-7 out of a total of six were removed, thus exposing an equivalent number of galactose residues at the terminal positions) by neuraminidase, injection into rats of this material resulted in a rapid and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver. The rate of this rapid transfer depended on the presence of intact galactose residues as the terminal, nonreducing sugar in the carbohydrate units. Galactose oxidase treatment of the partially desialylated (NANA-7)-α₂-AT, which presumably oxidized the primary alcohol of galactose at C-6 to an aldehyde group, caused a reversion of its survival time in the circulation to that of the intact (NANA-7)-α₁-AT.     

Viable nonsense mutants for the <Emphasis Type="Italic">SUP45</Emphasis> gene in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination factor eRF1 in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a trucated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37°C. When grown under nonpermissive conditions (37°C), cells of nonsense mutants sup45-104, sup45-105, and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA_gin and the other an amber suppressor with an altered tRNA_ser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.     

In vitro inhibition of mumps virus by retinoids

Background

Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules α_vβ₁, α_vβ₃, α_vβ₆, or α_vβ₈ by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the α_vβ₆ interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted α_vβ₆ (ssα_vβ₆) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction.

Results

Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssα_vβ₆. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssα_vβ₆ neutralization, relative ability to utilize α_vβ₆ for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for α_vβ₆. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssα_vβ₆. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssα_vβ₆ resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding.

Conclusions

These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.     

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.     

Intra- and extracellular forms of alpha-glucosidase from Aspergillus niger.

An intracellular α-glucosidase (α-glu₁) of Aspergillus niger was purified and its properties were compared to those of a secreted α-glucosidase (α-glu_E). The estimated molecular weight of α-glu_I was 95,000 by gel filtration (α-glu_E = 63,000); it is a glycoprotein possessing 29 mol of mannose, 6 mol of glucosamine, and 14 mol of glucose (α-glu_E has 5–6 and 2 mol of mannose and glucosamine, respectively). The K_m′s of α-glu₁ for p-nitrophenyl-α-d-glucopyranoside and maltose were 1.49 and 1.04, respectively, slightly lower than those of α-glu_E. In addition, at 65 °C α-glu_I enzymatic activity decayed fivefold faster than that of α-glu_E, and anti-α-glu_E antibody did not recognize α-glu_I. While some of these distinctions between the enzymes could be ascribed to conformational differences, the great dissimilarity in molecular weight (approximately 32,000) and lack of reactivity with anti-α-glu_E argue against α-glu_I being related to α-glu_E. The antibody covalently coupled to horseradish peroxidase (Ab-Px) was used as a probe to determine the cellular location of α-glu_E by electron microscopic immunocytology. It was found on both sides of the plasma membrane (pm) and in the outer of the two layers of the cell wall. This may mean that α-glu_E is synthesized at the inner surface of the pm, is extruded through the pm, becomes associated with the outer layer of the cell wall (perhaps as enzyme—substrate complex), and is eventually released into the growth medium.     

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

The mating type locus (MAT) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MATa and MATα, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MATα is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MATα. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, matα 1 mutants do not produce STE3 RNA, whereas matα 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MATα. Thus α1 positively regulates at least one α-specific gene by increasing the level of that gene's RNA product.     

Two mini-F-encoded proteins are essential for equipartition

The par region of mini-F is both necessary and sufficient to promote equipartition of plasmid copies to daughter cells. It is approximately 2.5 kb long and contains the coding sequences for two proteins, F1 (41 kDa) and F2 (37 kDa). We isolated 13 mutants of a phage λ-mini-F hybrid that form unstable plasmids. Two of these putative Par^? mutants are fully suppressible nonsense (amber) mutants. One of the amber mutants, par-41, eliminates the synthesis of F1, generating a large nonsense fragment of the protein. The other mutant, par-36, eliminates the synthesis of F2. Thus both proteins appear to be essential for plasmid equipartition.     

De novo synthesis of Escherichia coli glycogen is due to primer associated with glycogen synthase and activation by branching enzyme.

Previous reports have demonstrated the incorporation of glucose from ADP-glucose into methanol-insoluble and TCA-insoluble fractions in cell extracts of Escherichia coli in the absence of added primer α-glucan. This activity is reduced 6- to 76-fold in cell extracts of three independently isolated glycogen synthase-deficient mutants of E. coli B. Homogeneous preparations of E. coli B glycogen synthase catalyze incorporation of glucose into both methanol- and TCA-insoluble fractions in the absence of added primer. Since glycogen synthase catalyzes these reactions, it is not necessary to propose a protein acceptor glucose or a unique ADP-glucose-glycosyl transferase to catalyze formation of the glucoprotein in E. coli cell extracts to explain glucose incorporation into TCA-insoluble material (R. Barengo et al. (1975) FEBS Lett.53, 274–278). The incorporation of glucose into methanol-and TCA-insoluble fractions is stimulated by 0.25 m citrate and by branching enzyme. Citrate reduces the K_m for the primer, glycogen, about 11- to 15-fold. Branching enzyme can also reduce the concentration of primer required for incorporation of glucose into methanol-insoluble material. The simultaneous presence of both 0.25 m citrate and branching enzyme enables the glycogen synthase reaction rate to proceed at 30% the maximal velocity at a primer concentration of 1 μg/ml. Incorporation of glucose into methanol- or TCA-insoluble material in the absence of primer is completely inhibited by adding α-amylase. Furthermore, incorporation into methanol- or TCA-insoluble material is reduced 13- to 16-fold relative to the reaction occurring in the presence of primer when glycogen synthase is pretreated with glucoamylase and α-amylase. Previous results show that homogeneous preparations of glycogen synthase contain glucan. Heat-denatured glucogen synthase can act as a primer for the glycogen phosphorylase and glycogen synthase reactions. Both the TCA- and methanol-insoluble products form I₂-glucan complexes with wavelength maxima of about 580–590 nm and 610–615 nm, respectively, suggesting that they are mainly linear chain glucans. The products are completely solubilized with α-amylase. The TCA-insoluble product is not solubilized by pronase treatment. The above results strongly suggest that previous reports on formation of glucoprotein primer for glycogen synthesis or on de novo glycogen synthesis in various similar systems is due to endogenous glucan associated with glycogen synthase rather than formation of glucoprotein which then acts as primer for glycogen synthesis.     

Human-specific nonsense mutations identified by genome sequence comparisons

The comparative study of the human and chimpanzee genomes may shed light on the genetic ingredients for the evolution of the unique traits of humans. Here, we present a simple procedure to identify human-specific nonsense mutations that might have arisen since the human–chimpanzee divergence. The procedure involves collecting orthologous sequences in which a stop codon of the human sequence is aligned to a non-stop codon in the chimpanzee sequence and verifying that the latter is ancestral by finding homologs in other species without a stop codon. Using this procedure, we identify nine genes (CML2, FLJ14640, MT1L, NPPA, PDE3B, SERPINA13, TAP2, UIP1, and ZNF277) that would produce human-specific truncated proteins resulting in a loss or modification of the function. The premature terminations of CML2, MT1L, and SERPINA13 genes appear to abolish the original function of the encoded protein because the mutation removes a major part of the known active site in each case. The other six mutated genes are either known or presumed to produce functionally modified proteins. The mutations of five genes (CML2, FLJ14640, MT1L, NPPA, TAP2) are known or predicted to be polymorphic in humans. In these cases, the stop codon alleles are more prevalent than the ancestral allele, suggesting that the mutant alleles are approaching fixation since their emergence during the human evolution. The findings support the notion that functional modification or inactivation of genes by nonsense mutation is a part of the process of adaptive evolution and acquisition of species-specific features. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Radioactive labeling of alpha1-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives.

Human plasma α₁-antitrypsin (α₁-AT), bovine trypsin, and α-chymotrypsin were labeled with either ¹⁴C or ³H by reductive methylation. The labeled inhibitor retained the capacity to inactivate and to form 1:1 molar complexes with either the unlabeled or labeled trypsin and α-chymotrypsin. After intravenous injection of reductively methylated α₁-AT into rats, the labeled glycoprotein showed a circulating half-life of 12 h. When the N-acetylneuraminic acid residues were removed from the labeled α₁-AT by neuraminidase in vitro, injection into rats of this product resulted in a rapid (half-life of about 5 min) and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver of over 75% of the injected dose. The reductively methylated radioactively labeled trypsin and chymotrypsin experienced no loss of enzymatic activities. They showed the ability to form complexes in vivo with the two major plasma inhibitors, namely, α₁-AT and α₂-macroglobulin. High-voltage paper electrophoretic separation of acid hydrolysates of the labeled proteins revealed that ?-N-monomethyllysine and ?N,N-dimethyllysine are the only residues found to be radioactive.     

A rapid assay for the binding of actinomycin to DNA.

The theory of countercurrent distribution (CCD) was reviewed and extended. The separation function for the fundamental distribution of CCD was presented in the form n = t²(αk₁+β)²/αk₁(β?1)² where n is the number of transfers, t the abscissa of the standard normal distribution, α = v_m/v₈ the phase ratio, β = k₁/k₂≥ 1 the separation factor, and k₁ the partition coefficient of the more radidly moving component; n was found to be minimal on the condition αk₁ = β. The separation function for the single withdrawal of CCD was obtained in the form N = u + 1 = t²{(αk₁ + 1)^1/2 + [β(αk₁ + β)]^1/2}²/(β ? 1)²+ 1, where N is the number of partition units. From this equation it appears that N is minimal when αk₁ = 0. Compared with the former separation functions presented in the literature, these separation functions have the advantage of giving directly the relationships among the phase ratio, the absolute partition coefficient, the separation factor, the resolution degree, and the number of transfers or partition units required. In addition, the dependencies of the elution volumes and the widths of the elution curves on α, β, and the partition coefficients were considered mathematically by means of differential calculus. The elution volumes were found to have minima at certain αk₁ values. The standard deviations, on the contrary, did not have minima in respect to αk₁. The theory presented can be used for selecting proper operating conditions while separating chemical compounds.     

Glycosidases. Ligands for affinity chromotagraphy: II. Syntheses of p-aminophenyl 1-thio-L-fucopyranosides

Syntheses of p-aminophenyl 1-thio-α-L- and β-L-fucopyranosides are described. 1,2,3,4-Tetra-O-acetyl-α-L-fucopyranose, on heating with p-nitrothiophenol in the presence of p-toluenesulfonic acid under diminished pressure, gave a mixture of p-nitrophenyl 2,3,4-tri-O-acetyl-1-thio-α- and β-L-fucopyranosides, which was separated by chromatography on silica gel. When the reaction was carried out in the presence of zinc chloride at atmospheric pressure, the β-anomer was the exclusive product. Deacetylation of the aryl α-L- and α-L-thiofucopyranosides with sodium methoxide, followed by catalytic hydrogenation in the presence of palladium on barium sulfate, afforded the respective aminophenyl 1-thiofucopyranosides. The aryl thiofucopyranosides thus synthesized were tested for their inhibitory activity toward clam α-L-fucosidase. The p-aminophenyl 1-thio α-L-fucopyranoside showed a competitive-type inhibition, with a K_i of 0.71mM.