A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01–0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.

The dhp1+ gene, encoding a putative nuclear 5′→3′ exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1+ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)+ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1+ is required for proper chromosome segregation as well as for poly(A)+ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1+. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

A Gene Encoding a Hevein-Like Protein from Elderberry Fruits Is Homologous to PR-4 and Class V Chitinase Genes1

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene.

Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules1

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

The Schizosaccharomyces pombe spo20+ Gene Encoding a Homologue of Saccharomyces cerevisiae Sec14 Plays an Important Role in Forespore Membrane Formation

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+ is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20+ gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin

Zeatin is a naturally occurring cytokinin. Biosynthesis and metabolism studies of zeatin have been directed mostly at the trans isomer, although cis-zeatin and its riboside occur as major components in some plant species. It is not known whether parallel regulatory pathways exist for the two isomers. Based on the sequence of the gene ZOG1 encoding a trans-zeatin O-glucosyltransferase from Phaseolus (EC 2.4.1.203), a cis-zeatin-specific O-glucosyltransferase was isolated from maize. This gene, cisZOG1, contains an ORF of 1,401 nucleotides encoding a protein of 51.1 kDa with 41% identity to the Phaseolus ZOG1 protein. Unexpectedly, the maize enzyme recognizes as substrates cis-zeatin and UDP-glucose but not cis-ribosylzeatin, trans-zeatin, or trans-ribosylzeatin. This finding indicates the existence of cis-specific regulatory elements in plants and suggests that cis-zeatin and derivatives may be more important in cytokinin homeostasis than currently recognized.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

A Gene Encoding Proline Dehydrogenase Is Not Only Induced by Proline and Hypoosmolarity, but Is Also Developmentally Regulated in the Reproductive Organs of Arabidopsis1

The cDNA clone ERD5 (early responsive to dehydration), isolated from 1-h-dehydrated Arabidopsis, encodes a precursor of proline (Pro) dehydrogenase (ProDH), which is a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The transcript of the erd5 (ProDH) gene was undetectable when plants were dehydrated, but large amounts of transcript accumulated when plants were subsequently rehydrated. Accumulation of the transcript was also observed in plants that had been incubated under hypoosmotic conditions in media that contained l- or d-Pro. We isolated a 1.4-kb DNA fragment of the putative promoter region of the ProDH gene. The β-glucuronidase (GUS) reporter gene driven by the 1.4-kb ProDH promoter was induced not only by rehydration but also by hypoosmolarity and l- and d-Pro at significant levels in transgenic Arabidopsis plants. The promoter of the ProDH gene directs strong GUS activity in reproductive organs such as pollen and pistils and in the seeds of the transgenic plants. GUS activity was detected in vegetative tissues such as veins of leaves and root tips when the transgenic plants were exposed to hypoosmolarity and Pro solutions. GUS activity increased during germination of the transgenic plants under hypoosmolarity. The relationship between Pro metabolism and the physiological aspects of stress response and development are discussed.

Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.

Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible1

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species1

The C4 enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C4 plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C4 photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C4 cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C4 species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C4 form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.

Identification of the Gene Encoding the Tryptophan Synthase β-Subunit from Chlamydomonas reinhardtii1

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the β-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.