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1.
Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast 总被引:5,自引:0,他引:5
A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase. 相似文献
2.
Keinosuke Oda Haruyuki Atomi Mitsuyoshi Ueda Jun Kondo Yutaka Teranishi Atsuo Tanaka 《Archives of microbiology》1991,156(6):439-443
The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL
isocitrate lyase
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis 相似文献
3.
Anjali Madhavan Sriappareddy Tamalampudi Kazunari Ushida Daisuke Kanai Satoshi Katahira Aradhana Srivastava Hideki Fukuda Virendra S. Bisaria Akihiko Kondo 《Applied microbiology and biotechnology》2009,82(6):1067-1078
The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in
Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family
II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular
mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the
enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of
the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose. 相似文献
4.
REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end
of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron
in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice
site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of
yeast contain an intron at the same location in their REC114 genes.
Received: 16 October 1996 / Accepted: 10 February 1997 相似文献
5.
P. H. Benetti S. I. Kim M. Canonge T. Chardot J. C. Meunier 《Molecular & general genetics : MGG》1997,256(4):355-364
Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit
of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron
was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity
with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic
subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.
Received: 20 December1997 / Accepted: 19 June 1997 相似文献
6.
Cloning of Arabidopsis thaliana phosphatidylinositol synthase and functional expression in the yeast pis mutant 总被引:1,自引:0,他引:1
It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg2+ ions for full activity. 相似文献
7.
Haruyuki Atomi Ken Umemura Takanori Higashijima Tamotsu Kanai Yoshihisa Yotsumoto Yutaka Teranishi Mitsuyoshi Ueda Atsuo Tanaka 《Archives of microbiology》1995,163(5):322-328
The upstream region of the isocitrate lyase gene (UPR-ICL, 1530bp) of an n-alkane-utilizable yeast, Candida tropicalis, induced gene expression in another yeast, Saccharomyces cerevisiae, when the yeasts were grown on acetate. Surprisingly, UPR-ICL displayed the same regulatory function in the bacterium Escherichia coli when grown on acetate. We determined the interesting nucleotide sequence of UPR-ICL. The deletion analysis of UPR-ICL in both cells revealed the presence of two distinct promoters: one was localized at-394 to-379 and regulated gene expression in S. cerevisiae; the other was tocated near the initiation codon and regulated gene expression in E. coli. The two promoter sequences were similar, but not identical to regulatory elements that have been previously reported in S. cerevisiae and E. coli, respectively. Accordingly, the possibility of novel regulatory mechanisms could not be excluded. This is an interesting example of the presence of distinct cis-acting regulatory elements responsible for the induction of gene expression in one gene by acetate in both S. cerevisiae and E. coli. Preservation of such promoters through evolution is also discussed.Abbreviations
ICL
Isocitrate lyase
-
UPR-ICL
Upstream region of the Candida tropicalis isocitrate lyase gene 相似文献
8.
Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae 总被引:5,自引:0,他引:5
Lindahl AL Olsson ME Mercke P Tollbom O Schelin J Brodelius M Brodelius PE 《Biotechnology letters》2006,28(8):571-580
The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression
vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the
yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed
for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while
plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced
600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation.
Revisions requested 14 November 2005; Revisions received 17 January 2006 相似文献
9.
We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-μm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment
revealed that the ARS-acting sequence was located in the 3′-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae.
Received: 16 June 1998 / Accepted: 3 August 1998 相似文献
10.
A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis> 总被引:3,自引:0,他引:3
Yu P 《Applied microbiology and biotechnology》2007,74(1):93-98
The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme
SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The
SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column
chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity. 相似文献
11.
The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0
kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence
of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 %
glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase
activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted
α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture
filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364. 相似文献
12.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
13.
Toshiya Imajo Hiroyuki Kawachi Haruyuki Atomi Shin-ichi Sanuki Setsuko Yamamoto Mitsuyoshi Ueda A. Tanaka 《Archives of microbiology》1997,168(5):389-395
Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the
yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms.
The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported
Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular
mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial
Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence
of another gene responsible for peroxisomal Idp in C. tropicalis.
Received: 11 March 1997 / Accepted: 24 June 1997 相似文献
14.
S. Rahman Z. Li S. Abrahams D. Abbott R. Appels M. K. Morell 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(1):156-163
A genomic DNA fragment from Triticum tauschii, the donor of the wheat D genome, contains a starch branching enzyme-I (SBE-I) gene spread over 6.5 kb. This gene (designated
wSBE I-D4) encodes an amino acid sequence identical to that determined for the N-terminus of SBE-I from the hexaploid wheat (T. aestivum) endosperm. Cognate cDNA sequences for wSBE I-D4 were isolated from hexaploid wheat by hybridisation screening from an endosperm library and also by PCR. A contiguous sequence
(D4 cDNA) was assembled from the sequence of five overlapping partial cDNAs which spanned wSBE I-D4. D4 cDNA encodes a mature polypeptide of 87 kDa that shows 90% identity to SBE-I amino acid sequences from rice and maize and
contains all the residues considered essential for activity. D4 mRNA has been detected only in the endosperm and is at a maximum concentration mid-way through grain development. The wSBE I-D4 gene consists of 14 exons, similar to the structure for the equivalent gene in rice; the rice gene has a strikingly longer
intron 2. The 3′ end of wSBE I-D4 was used to show that the gene is located on group 7 chromosomes. The sequence upstream of wSBE I-D4 was analysed with respect to conserved motifs.
Received: 14 January 1998 / Accepted: 14 July 1998 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1580-1583
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene. 相似文献
16.
Ryuhei Funabiki Nobuhiro Horisaka Atutane Ohta 《Bioscience, biotechnology, and biochemistry》2013,77(6):1641-1642
Zygosaccharomyces rouxii contains at least two copies of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP-DH). We cloned one of them, which is situated on a 9.5 kb Hindlll fragment, after detecting a sequence which is hybridizable with the GAP-DH gene of Saccharomyces cerevisiae. The amino acid sequence of the GAP-DH gene of Z. roxii deduced from its nucleotide sequence was compared with that of the GAP-DH genes of S. cerevisiae. The Z. rouxii enzyme is longer than the S. cerevisiae enzyme by one amino acid. 65, 66 or 71 amino acid changes were detected, depending on which GAP-DH gene of S. cerevisiae was compared with that of the Z. rouxii enzyme. The codon usage in the coding region of the GAP-DH gene of Z. rouxii is biased, as found in the case of the S.cerevisiae enzyme. 相似文献
17.
Jones GW Hooley P Farrington SM Shawcross SG Iwanejko LA Strike P 《Molecular & general genetics : MGG》1999,261(2):251-258
Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency. The
sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone. Initial sequencing of
a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show
that this DNA is transcribed. A 277-bp fragment derived from the transcribed region was used to screen an A. nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment. This partial cDNA was incomplete but appeared to contain the whole coding region of sagA. The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+1 frameshift due to insertion of a T, causing disruption
to the C-terminal region of the SagA protein. Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation.
Received: 1 August 1998 / Accepted: 9 November 1998 相似文献
18.
Katsuyama Y Miyahisa I Funa N Horinouchi S 《Applied microbiology and biotechnology》2007,73(5):1143-1149
For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml
of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the
purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete
Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium
glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S.
cerevisiae cell containing the IFS gene. Coincubation of the E.
coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate
yielded ca. 6 mg genistein/l. 相似文献
19.
20.
Summary We report here the complete nucleotide sequence of the E. coli triose phosphate isomerase gene. The gene encodes a polypeptide of 255 amino acids which is approximately 46% homologous to eukaryotic triose phosphate isomerases, and approximately 38% homologous to the enzyme from a thermophilic bacterium, Bacillus stearothermophilus. The nucleotide sequence is 55% homologous to that of the corresponding gene in the yeast Saccharomyces cerevisiae. To our knowledge, this is the first report of the sequence of a gene coding a glycolytic enzyme from a prokaryotic organism. 相似文献