Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39°C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 106 mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Adaptor Complex-independent Clathrin Function in Yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

Genetic and Biochemical Characterization of the Yeast Spo12 Protein

We have performed a genetic and biochemical analysis of the SPO12 gene, which regulates meiotic nuclear divisions in budding yeast. When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II. We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation. Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p. We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, and SPO13, whose mutant phenotype is like that of spo12, can partially suppress the meiotic defect of spo12 mutants when overexpressed. We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M. We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation in HCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.

Isolation and characterization of a pseudoautosomal region-specific genetic marker in C57BL/6 mice using genomic representational difference analysis.

Representational difference analysis was used to identify strain-specific differences in the pseudoautosomal region (PAR) of mouse X and Y chromosomes. One second generation (C57BL/6 x Mus spretus) x Mus spretus interspecific backcross male carrying the C57BL/6 (B6) PAR was used for tester DNA. DNA from five backcross males from the same generation that were M. spretus-type for the PAR was pooled for the driver. A cloned probe designated B6-38 was recovered that is B6-specific in Southern analysis. Analysis of genomic DNA from several inbred strains of laboratory mice and diverse Mus species and subspecies identified a characteristic Pst I pattern of fragment sizes that is present only in the C57BL family of strains. Hybridization was observed with sequences in DBA/2J and to a limited extent with Mus musculus (PWK strain) and Mus castaneus DNA. No hybridization was observed in DNA of different Mus species, M. spretus, M. hortulanus, and M. caroli. Genetic analyses of B6-38 was conducted using C57BL congenic males that carry M. spretus alleles for distal X chromosome loci and the PAR and outcrosses of heterozygous congenic females with M. spretus. These analyses demonstrated that the B6-38 sequences were inherited with both the X and Y chromosome. B6-38 sequences were genetically mapped as a locus within the PAR using two interspecific backcrosses. The locus defined by B6-38 is designated DXYRp1. Preliminary analyses of recombination between the distal X chromosome gene amelogenin (Amg) and the PAR loci for either TelXY or sex chromosome association (Sxa) suggest that the locus DXYRp1 maps to the distal portion of the PAR.

Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of nonmotile mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nonmotile mutant of F. johnsoniae (UW102-09) with a library of wild-type DNA by using the shuttle cosmid pCP17. The complementing plasmid (pCP100) contained an insert of 13 kbp, and restored motility to 4 of 61 independently isolated nonmotile mutants. A 1.3-kbp fragment that encompassed a single ORF, gldA, complemented all four mutants. Disruption of the chromosomal copy of gldA in wild-type F. johnsoniae UW101 eliminated gliding motility. The predicted protein produced by gldA has strong sequence similarity to ATP binding cassette transport proteins.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

Human sulfite oxidase R160Q: Identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.