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Cloning a segment of the gene encoding 3-hydroxy-3-methylglutaryl Coenzyme A reductase in Phycomyces blakesleeanus and Gibberella fujikuroi by the polymerase chain-reaction
Abstract
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase catalyzes the synthesis of mevalonate, the precursor of all isoprenoid compounds in plants and animals. We have amplified, cloned, and sequenced a segment of the gene encoding the HMG-CoA reductase (hmgA) from the fungi Phycomyces biakesleeanus and Gibberella fujikuroi. The DNA amplification was performed with degenerate primers designed to anneal at conserved regions of the gene separated by about 300 bp. The sequences can now be used as probes to isolate the complete genes and to study their expression. The same protocol should allow the amplification of the homologous DNA segment from the genome of any other eukaryote. This is the first report of a DNA sequence from G. fujikuroi.
... is the key enzyme in biosynthesis of polyisoprenoids [5]. The gene could be isolated from several mammalia [6- [20] the genes were isolated and partially characterized. Whereas the 5' termini of the genes differ considerably from each other, the 3' part is well conserved between all eukaryotic sequences [ 161. ...
... The polymerase chain reaction (PCR) was performed with two degenerate primers, similar to the primers for isolating the Phycomyces blakesleeanus segment [20]. The sequences of the primers were 5' GgaattcCGATGGCGAC-GACGGAAGG 3' for the 5' end and 5' GGaagcttCATGTT-CATGCCCAT 3' for the 3' end of the fragment. ...
... The HMG2 genes from both M. mucedo and f! parasitica were also highly similar, whereas the gene from A. glauca is less similar. Comparison of the gene from A. glauca with the other genes from zygomycetes shows the highest similarity between A. glauca and Phycomyces blakesleeanus [20]. The percentage identity of amino acid positions of the characterized sequences, including the l? blakesleeanus sequence [20], are summarized in detected between the HMGl and HMG2 gene from one species. ...
In this paper we compare the sequences of a segment of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene, isolated from eleven different strains belonging to four species of the fungal order Mucorales, Parasitella parasitica, Absidia glauca, Mucor mucedo (Mucoraceae) and Blakeslea trispora (Choanephoraceae). The segment was obtained by polynucleotide-chain-reaction amplification with primers derived from conservative regions of the gene. For the species M. mucedo and P. parasitica we have obtained evidence for two different types of HMG-CoA reductase genes by hybridization of genomic DNA with the amplified fragment and by cloning and sequencing of two different fragments. The different genes from one species show a sequence similarity of around 80% at the protein sequence level, whereas sequences of the same type from different species show similarity ranging between 91-96%. The highest similarity was found between the genes of type 1 from B. trispora and M. mucedo, although these species belong to different families. Southern-blot analysis of A. glauca DNA and B. trispora DNA revealed a second copy of the genes.
... Indeed, HMGR enzyme tests were negative. In addition, we failed to amplify DNA from S. multivorum by using HMGR-speci¢c probes recognizing the genes from many organisms [21], while ampli¢cation was possible from tobacco BY-2 cells and from Saccharomyces cerevisiae, organisms where the presence of the corresponding genes was proved earlier. The degenerate primers (forward : 5P-CCI ATG ACI GA(AG) GG-3P; reverse: 5P-CAT (GA)TT CAT ICC CAT-3P), designed from two highly conserved sequences £anking amino acids 260^365 in the Arabidopsis thaliana HMGR protein [22] (revised SwissProt accession number P14891), amplify a fragment of about 300 bp under the used PCR conditions [21]. ...
... In addition, we failed to amplify DNA from S. multivorum by using HMGR-speci¢c probes recognizing the genes from many organisms [21], while ampli¢cation was possible from tobacco BY-2 cells and from Saccharomyces cerevisiae, organisms where the presence of the corresponding genes was proved earlier. The degenerate primers (forward : 5P-CCI ATG ACI GA(AG) GG-3P; reverse: 5P-CAT (GA)TT CAT ICC CAT-3P), designed from two highly conserved sequences £anking amino acids 260^365 in the Arabidopsis thaliana HMGR protein [22] (revised SwissProt accession number P14891), amplify a fragment of about 300 bp under the used PCR conditions [21]. ...
Feeding of [1-13C]glucose, [U-13C6]glucose, [3-13C]alanine and [1-13C]acetate to Sphingobacterium multivorum showed that this bacterium utilizes the methylerythritol phosphate pathway for the biosynthesis of menaquinone-7 and zeaxanthin, a carotenoid of industrial importance. Differential incorporation of the labeled precursors gave some insight into the preferred carbon sources involved in isoprenoid biosynthesis. © 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
... t Permanent address: Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary. phytopathological research to detect plant pathogenic fungi in host tissues directly (5,13,26,30,33) and to isolate a fungal gene sequence relevant in terpenoid biosynthesis (4). ...
... t Permanent address: Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary. phytopathological research to detect plant pathogenic fungi in host tissues directly (5,13,26,30,33) and to isolate a fungal gene sequence relevant in terpenoid biosynthesis (4). ...
We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.
... The terpenoids include the carotenoids, the sterols, the dolichols, and various hormones, lipid moieties in membrane proteins, ubiquinone and chlorophyll (Conolly and Hill 1992). Many organisms synthesize the early precursors of the terpenoids by condensing acetyl coenzyme A (acetyl-CoA) with acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), and converting this intermediate to mevalonate. ...
Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids. The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and its conversion to mevalonate. We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus. Single copies of these genes are present in the Phycomyces genome. The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain. The hydrophilic domain suffices for catalytic activity, as shown by expressing it in Escherichia coli. Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing. Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMG-CoA.
Carotenoids occur universally in photosynthetic organisms but sporadically in nonphotosynthetic bacteria and eukaryotes. The
primordial carotenogenic organisms were cyanobacteria, and eubacteria that carried out anoxygenic photosynthesis. The phylogeny
of carotenogenic organisms is evaluated to describe groups of organism which could serve as sources of carotenoids. Terrestrial
plants, green algae, and red algae acquired stable endosymbionts (probably cyanobacteria) and have a predictable complement
of carotenoids compared to prokaryotes, other algae, and higher fungi which have a more diverse array of pigments. Although
carotenoids are not synthesized by animals, they are becoming known for their important role in protecting against damage
by singlet oxygen and preventing chronic, diseases in humans. The growth of aquaculture during the past decade as well as
the biological roles of carotenoids in human disease will increase the demand for carotenoids. Microbial synthesis offers
a promising method for production of carotenoids.
The rich metabolism of the fungal strains, now classified as Fusarium fujikuroi and related species covers the production of many secondary products, including different terpenoids and polyketides. Research has concentrated on the gibberellins, growth-promoting plant hormones with multiple applications in agriculture and brewing. The long list of secondary metabolites includes pigments such as carotenoids and bikaverins, mycotoxins such as fusarins and fumonisins and a diversity of other compounds, many of them still being unidentified. This review describes the chemical structures of the secondary metabolites identified in these fungi, with special attention to the terpenoids and particularly to the gibberellins, and summarizes knowledge on the biosynthesis of gibberellins, its regulation and its relation to those of other metabolites.
In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor
hmgR did not affect the carotenoid production of M. circinelloides.
Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development.
The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.
The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G + C) content, 35%; the coding sequences cloned to date all have a G + C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5'-CCGG-3') and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G + C-rich genes surrounded by G + C-poor areas. Two clones have similarities to the transposase of the transposon Tc1 from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tc1-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Tx1 from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 bp named PrA1 and PrA2.
The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.
Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate in different subcellular compartments of the fungus Gibberella fujikuroi. Lovastatin inhibits growth in many organisms, presumably because of the inhibition of the synthesis of essential terpenoids. However, in G. fujikuroi growth of the mycelia and sterol and carotenoid content were not affected by the presence of lovastatin. Nevertheless, lovastatin did inhibit the accumulation of gibberellins in the culture medium; this inhibition, however, was counteracted by the addition of mevalonate to the medium. The conversion of HMG-CoA to mevalonate in cell-free extracts was inhibited by 10 nM lovastatin. Since G. fujikuroi apparently possesses a single gene for HMG-CoA reductase, as shown by Southern hybridization and PCR amplification, it was concluded that the biosynthesis of sterols, carotenoids and gibberellins shares a single HMG-CoA reductase, but the respective subcellular compartments are differentially accessible to lovastatin.
The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. We have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, we have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, we have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, we have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.
We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements trpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpC- strain to trpC+ at frequencies of greater than 20 stable transformants per microgram of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the trpC- allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.
Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m(-2) blue light and produces a large beta-carotene accumulation, which is not saturated at 2·10(6) J·m(-2). Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.
The orange pigment neurosporaxanthin colours the mycelia of wild Gibberella fujijuori (Fusarium monifliforme) grown in the light, but is barely detectable in the dark. We have isolated carotenoid mutants from conidia exposed to N-methyl-N-nitro-N-nitroso-guanidine and other mutagens. Specific blocks in the pathway are represented by white mutants accumulating phytoene and red mutants accumulating torulene; there are also mutants without carotenoids or with complex carotenoid mixtures. Regulatory mutants overproduce neurosporaxanthin, both in the light and in the dark. Other mutants contain considerable neurosporaxanthin in the dark, but less than in the light. The results bring out similarities between the carotenoid biosynthetic pathways of Gibberella and Phycomyces, and significant differences in their respective regulations.
A strategy employing the polymerase chain reaction to synthesize gene-specific probes suitable for genomic Southern analyses and for screening genomic libraries is described. The method utilizes partial amino acid sequence data from the protein of interest, genomic DNA and inosine-containing oligonucleotide primers. An example of its application for the isolation of plant gene sequences encoding saporin, a ribosome inactivating protein, is described.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) catalyses the synthesis of mevalonate, the specific precursor of all isoprenoid compounds present in plants. We have characterized two overlapping cDNA clones that encompass the entire transcription unit of an HMG-CoA reductase gene from Arabidopsis thaliana. The transcription product has an upstream non-coding sequence of 70 nucleotides preceding an open reading frame of 1776 bases and a 3' untranslated region in which two alternative polyadenylation sites have been found. The analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 592 residues with a molecular mass of 63,605 Da. The hydropathy profile of the protein indicates the presence of two highly hydrophobic domains near the N-terminus. A sequence of 407 amino acids corresponding to the C-terminal part of the protein (residues 172-579), which presumably contains the catalytic site, shows a high level of similarity to the region containing the catalytic site of the hamster, human, yeast and Drosophila enzymes. The N-terminal domain contains two putative membrane-spanning regions, in contrast to the enzyme from other organisms which has seven trans-membrane regions. A. thaliana contains two different HMG-CoA reductase genes (HMG1 and HMG2), as estimated by gene cloning and Southern blot analysis. Northern blot analysis reveals a single transcript of 2.4 kb in leaves and seedlings, which presumably corresponds to the expression of the HMG1 gene.
Regulation of HMG-CoA Reductase P~mer-directed enzymatic amplification of DNA with a thermo-stable DNA polymerase Transformation of Aspergillus niduians by using a trpC plasmid
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