Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity.

To obtain large quantities of pure human beta 2-adrenergic receptor (beta 2-AR) needed for structural studies, an efficient method for beta 2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged beta 2-AR was expressed in Sf9 cells with a specific activity of 5-20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-beta-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60-70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of > 30%. The purified receptor was concentrated to > 1 mg/ml without loss of ligand binding activity.

The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein.

The GCR1 gene product is required for maximal transcription of yeast glycolytic genes and for growth of yeast strains in media containing glucose as a carbon source. Dominant mutations in two genes, SGC1 and SGC2, as well as recessive mutations in the SGC5 gene were identified as suppressors of the growth and transcriptional defects caused by a gcr1 null mutation. The wild-type and mutant alleles of SGC1 were cloned and sequenced. The predicted amino acid sequence of the SGC1 gene product includes a region with substantial similarity to the basic-helix-loop-helix domain of the Myc family of DNA-binding proteins. The SGC1-1 dominant mutant allele contained a substitution of glutamine for a highly conserved glutamic acid residue within the putative basic DNA binding domain. A second dominant mutant, SGC1-2, contained a valine-for-isoleucine substitution within the putative loop region. The SGC1-1 dominant mutant suppressed the GCR1 requirement for enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase gene expression. Expression of the yeast enolase genes was reduced three- to fivefold in strains carrying an sgc1 null mutation, demonstrating that SGC1 is required for maximal enolase gene expression. Expression of the enolase genes in strains carrying gcr1 and sgc1 double null mutations was substantially less than observed for strains carrying either null mutation alone, suggesting that GCR1 and SGC1 function on parallel pathways to activate yeast glycolytic gene expression.

The Escherichia coli hflA locus encodes a putative GTP-binding protein and two membrane proteins, one of which contains a protease-like domain.

The hflA (high frequency of lysogenization) locus of Escherichia coli governs the lysis-lysogeny decision of bacteriophage lambda by controlling stability of the phage cII protein. hflA contains three genes, hflX, hflK, and hflC, encoding polypeptides of 50, 46, and 37 kDa, respectively. We have determined the nucleotide sequence of 3843 base pairs containing hflA and have found three large open reading frames corresponding to hflX, hflK, and hflC. HflX contains the three sequence motifs typical of GTP-binding proteins and appears to be a member of a distinct family of putative GTPases. HflC and HflK appear to be integral membrane proteins which show some similarity to each other and to a human membrane protein. The C-terminal region of HflC contains a domain resembling the catalytic domain of ClpP, a bacterial ATP-dependent protease. We hypothesize that HflK and HflC constitute a distinct membrane-bound protease whose activity may be modulated by HflX GTPase.

Phenotypic analysis of proteinase A mutants. Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae.

We have isolated a number of mutants deficient in activity of the vacuolar hydrolase proteinase A (PrA). The mutations were sequenced and although they all map in the PEP4 gene, which encodes the precursor to PrA, three distinguishable phenotypes have surfaced. The properties of the pep4-7 missense mutant suggested that the activation of the precursor to proteinase A is due to an autocatalytic cleavage. PrA active site mutations were constructed and resulted in accumulation of PrA antigen in the inactive precursor form. Although protease B (PrB), another vacuolar hydrolase, is not required for the production of active PrA, the active form of PrA that accumulates in a strain lacking PrB is larger than that found in a strain containing active PrB. We have purified this larger form of PrA and determined that it bears 7 additional amino acids at its NH2 terminus. It has become apparent from all the studies performed on the maturation pathway of the vacuolar hydrolases that there is a great deal of redundancy built into the system.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.

Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2: ABFI protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription.

Binding sites for three distinct proteins were mapped within the upstream activation sites (UAS) of the yeast enolase genes ENO1 and ENO2. Sequences that overlapped the UAS1 elements of both enolase genes bound a protein which was identified as the product of the RAP1 regulatory gene. Sequences within the UAS2 element of the ENO2 gene bound a second protein which corresponded to the ABFI (autonomously replicating sequence-binding factor) protein. A protein designated EBF1 (enolase-binding factor) bound to sequences which overlapped the UAS2 element in ENO1. There was a good correlation among all of the factor-binding sites and the location of sequences required for UAS activity identified by deletion mapping analysis. This observation suggested that the three factors play a role in transcriptional activation of the enolase genes. UAS elements which bound the RAP1 protein or the ABFI protein modulated glucose-dependent induction of ENO1 and ENO2 expression. The ABFI-binding site in ENO2 overlapped sequences required for UAS2 activity in wild-type strains and for repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1. These latter results showed that the ABFI protein, like the RAP1 protein, bound sequences required for positive as well as negative regulation of gene expression. These observations strongly suggest that the biological functions of the RAP1 and ABFI proteins are similar.

Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans.

Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis. This gene cluster is required for the assimilation of nitrate in A. nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively. The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed. Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively. The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium. Two types of transcripts for niiA were observed.

Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu.

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.

DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene.

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.

We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent.

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

The filamentous ascomycete Aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. We examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mRNA in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. We postulate that induction of glucoamylase synthesis is regulated transcriptionally. Comparing total mRNA from cells grown on starch and xylose, we were able to identify an inducible 2.3-kilobase mRNA-encoding glucoamylase. The glucoamylase mRNA was purified and used to identify a molecularly cloned 3.4-kilobase EcoRI fragment containing the A. awamori glucoamylase gene. Comparison of the nucleotide sequence of the 3.4-kilobase EcoRI fragment with that of the glucoamylase I mRNA (as determined from molecularly cloned cDNA) revealed the existence of four intervening sequences within the glucoamylase gene. The 5' end of the glucoamylase mRNA was mapped to several locations within a region -52 to -73 nucleotides from the translational start. Sequence and structural features of the glucoamylase gene of the filamentous ascomycete A. awamori were examined and compared with those reported in genes of other eucaryotes.

Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene.

The nucleotide sequence of the prolipoprotein signal peptidase (lsp) gene has been determined. The lsp gene was found to be adjacent to the isoleucyl-tRNA synthetase ( ileS ) gene, such that the termination codon of the ileS gene overlaps with the initiation codon of lsp. These two genes are transcribed in the same direction and the major promotor for the lsp gene appears to be upstream of ileS . Identification of the lsp gene was established by amplification of prolipoprotein signal peptidase activity in strains carrying a subcloned 1.1-kilobase Stu I-Acc I fragment and was further confirmed by introducing mutational alterations in the COOH terminus of the protein that caused a decrease in prolipoprotein signal peptidase activity. The deduced amino acid sequence indicates that prolipoprotein signal peptidase contains 164 residues. Unlike most exported proteins, there is no apparent signal peptide sequence for the lsp protein. Computer-assisted secondary structure analysis of the deduced amino acid sequence identified four hydrophobic regions that share features common to transmembrane segments in integral membrane proteins.