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1.
A specific endonuclease from Arthrobacter luteus.   总被引:20,自引:0,他引:20  
A new restriction-like endonuclease, AluI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage λ DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the endonuclease HindIII from Haemophilus influenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5′-32P)-labelled fragments of phage λ DNA released by the action of AluI had the 5′ terminal sequence pC-T-N-. The enzyme recognises the tetranucleotide sequence
and cleaves it at the position marked by the arrows.  相似文献   

2.
An endonuelease R.HindIII, prepared from Hemophilus influenzae strain Rd, degrades foreign DNA, but not homologous DNA. Phage T7 DNA is also resistant to the enzyme. Fragments of phage λ DNA produced by treatment with R.HindIII have been labelled at their 5′ termini and analysis of the radioactive nucleotides in pancreatic DNAase digests of these fragments revealed a single 5′ terminal sequence. From this and other data we conclude that the enzyme recognizes and cleaves DNA at the following nucleotide sequence,
giving termini bearing short cohesive ends.  相似文献   

3.
The DNA sequence recognised by the HinfIII restriction endonuclease   总被引:3,自引:0,他引:3  
HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:
5′-C-G-A-A-T-3′·····3′-G-C-T-T-A-5′
In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.  相似文献   

4.
Restriction endonuclease EcoRI cuts both strands of the DNA sequence
generating two separate frayed ends (Hedgpeth et al., 1972). Here it is shown that under standard digestion conditions, the enzyme also attacks the sequence
but cuts only one strand. The resulting nick is an efficient initiation point for DNA synthesis by Escherichia coli DNA polymerase I, allowing the selective labelling of one strand of the DNA duplex.In buffers of low molarity and high pH (8.5), EcoRI cleaves sequences with the form
(Polisky et al., 1975). Thus it seems that under both sets of conditions the enzyme recognises the four-base-pair core sequence
and that its ability to cleave different adjacent phosphodiester bonds varies with pH and ionic strength.  相似文献   

5.
A new specific endonuclease from Xanthomonas badrii   总被引:12,自引:0,他引:12  
A restriction-like endonuelease, XbaI, has been partially purified from Xanthomonas badrii. This enzyme cleaves adenovirus-2 DNA at four sites, bacteriophage lambda DNA at only one site, and does not cleave simian virus 40 DNA. It recognizes the sequence
and cuts at the sites indicated by the arrows.  相似文献   

6.
7.
DNA methylation in Bacillus amyloliquefaciens strain H (Bam)2 and Bacillus brevis (Bbv) has been examined by a variety of techniques. In vivo labelling studies revealed that Bam DNA contains no N6-methyladenine (MeAde), but contains 5-methylcytosine (MeCyt); approximately 0·7% of the cytosine residues are methylated.DNA methylase activity was partially purified from both Bam and Bbv; the Bam enzyme preparation transferred methyl groups from S-adenosyl-l-[methyl-3H]methionine ([3H]AdoMet) to specific DNA cytosine residues only; in agreement with Vanyushin & Dobritsa (1975), the Bbv enzyme preparation methylated both DNA adenine and cytosine residues. The (partial) sequence specificity of the methylases was determined by analyzing [3H]methyl-labelled dinucleotides obtained from enzymatic digests of DNA methylated in vitro. Bam and Bbv each contain a DNA-cytosine methylase with overlapping sequence specificity; e.g. both enzymes produce G-C1, C1-A and C1-T. This is consistent with a single, twofold symmetrical methylation sequence of 5′ … G-C1-(A or T)-G-C … 3′; this was observed by Vanyushin & Dobritsa (1975) for a different Bbv strain. Bam contains a second DNA-cytosine methylase (not present in Bbv), which produces T-C1 and C1-T. We propose that this methylase is the BamI modification enzyme, and that the modified sequence is 5′ … G-G-A-T-C1-C … 3′.Bbv appears to contain two DNA-adenine methylases which produce the (partial) methylated sequences, 5′ … G-A1-T … 3′ and 5′ … A-A1-G … 3′, respectively; in the former case, all the G-A-T-C sites on Bbv DNA appear to be methylated.  相似文献   

8.
9.
The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5′-GAGTC-3′) includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5′-GAGTC-3′ and was specific for 5′-GASTC-3′ substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min?1, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM.  相似文献   

10.
11.
12.
A new restriction endonuclease from Streptomyces albus G.   总被引:10,自引:0,他引:10  
A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence
and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.  相似文献   

13.
A new specific endonuclease, XhoI, has been partially purified from Xanthomonas holcicola. This enzyme cleaves adenovirus-2 DNA at five sites, bacteriophage λ DNA at one site, φX174 DNA at one site, but does not cleave simian virus 40 DNA. It recognizes the sequence
and cuts at the sites indicated by the arrows. Enzymes with identical specificity have also been found in Xanthomonas papavericola and Brevibacterium luteum.  相似文献   

14.
15.
16.
17.
pSTNV-1 is a chimera plasmid that contains a nearly full-size double-stranded DNA copy of the satellite tobacco necrosis virus RNA genome (see preceding paper by van Emmelo et al., 1980) and we report here the complete nucleotide sequence of this STNV2 DNA insert. The results show that except for 23 nucleotide pairs corresponding to the 5′ end of STNV RNA, a full-size STNV DNA copy is present in pSTNV-1. The total nucleotide sequence of the STNV genome contains 1239 residues. The amino acid sequence of the coat protein can be deduced from the 5′ half of the DNA message strand and shows a rather hydrophobic carboxyl-terminal region and a basic amino-terminal region. The 3′ untranslated part of the viral RNA is 622 nucleotides long. A secondary structure model for the 5′ end showing an interaction with a segment in the 3′ half is proposed. The 3′ end region can be folded into a transfer RNA cloverleaf-like structure with an anticodon for AUG.  相似文献   

18.
19.
In this study, the atp8 gene was cloned from the cytoplasmic male sterile (CMS) line UG93A and its maintainer line UG93B in kenaf. Its DNA sequence analysis showed that atp8 containing 480-bp, encoding 159 amino acid residues, and a 9-bp insertion was found at the 3′flanking sequence in UG93A compared with UG93B. The cDNA sequence of atp8 analyzed by RT-PCR indicated that there were five loci edited, but six loci edited in UG93B. The editing frequencies were higher in sterile cytoplasm than in fertile cytoplasm. The relative expression of atp8 analyzed by real-time PCR showed that the expressed level of atp8 in UG93A was lower than that of its maitainer UG93B and its F1 hybrid UG93A/992 (a restore line). Furthermore, based on the difference of the 9-bp differences at the 3′flanking sequence of atp8 between UG93A and UG93B, a molecular marker specific to male sterile cytoplasm was developed, which can be used for indentifying whether any germplasm of kenaf is male sterile cytoplasm or male fertile cytoplasm.  相似文献   

20.
The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of l-idonic acid in which d-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes l-idonate 5-dehydrogenase, 5-keto-d-gluconate 5-reductase, an l-idonate transporter, and an l-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of l-idonate; IdnD catalyzes a reversible oxidation of l-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form d-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of d-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with l-idonate or 5-ketogluconate serving as the true inducer of the pathway. The l-idonate 5-dehydrogenase and 5-keto-d-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of l-idonate and d-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize l-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on l-idonate is altered.In Escherichia coli, the Entner-Doudoroff (ED) pathway serves as a metabolic “funnel” receiving intermediates formed by catabolism of several sugar acids (17). Hexuronic acids undergo rearrangement in the inducible Ashwell pathways (1) to form 2-keto-3-deoxygluconate, which is then phosphorylated to produce 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG is cleaved by KDPG aldolase, encoded by eda, providing for entry of carbon into glycolysis. The other enzyme of the ED pathway is 6-phosphogluconate dehydratase, encoded by edd, which is induced only for catabolism of gluconate and also forms KDPG, the key intermediate of the ED pathway (7). Long considered to be of more significance than is readily obvious (9), the finding that eda and edd eda double mutants are unable to colonize the mouse large intestine underscores the possible ecological importance of ED metabolism (32). The implication from these colonization studies is that colonic mucus, which contains several sugar acids, may serve as an important source of nutrients for E. coli in the gut.Also participating in gluconate catabolism are several gluconate transporters and two gluconate kinases which appear, based upon their regulation, to comprise two distinct systems (2, 13). The GntI (main) system consists of gntT, gntU, and gntK, which code for high- and low-affinity gluconate transporters and a thermoresistant gluconate kinase, respectively (2325, 33). Expression of the GntR regulon, that is, GntI together with the edd-eda operon, is negatively controlled by the gntR gene product. The GntII (subsidiary) system is comprised of a thermosensitive gluconate kinase and a gluconate transporter which function for gluconate catabolism in the absence of the GntI system (2, 11, 13, 22). It appears that the subsidiary gluconate transporter, which has an apparent Km for gluconate of 60 μM (23), is encoded by a gene (idnT) which is adjacent to the gene encoding the thermosensitive gluconokinase (idnK) at 96.8 min.The DNA sequence of the GntII system genes, located at 4492 kb on the genome, was revealed by the E. coli Genome Project (5, 6). If the GntII system had evolved as a subsidiary pathway for gluconate catabolism, one would expect it to contain only a gluconate transporter and gluconate kinase. However, in addition to the divergent idnK and idnT genes, this region also encodes two “dehydrogenase-like” enzymes. The similarity of idnO to gno of Gluconobacter oxydans, which encodes d-gluconate:NADP 5-oxidoreductase (GNO) (15), led to the testing of ketogluconates as enzyme substrates for the two newly identified dehydrogenases. A process of deductive reasoning and biochemical experiments led to the conclusion that the GntII system in fact comprises a novel metabolic pathway for catabolism of l-idonic acid, in which gluconate is a key intermediate. Accordingly, the genes involved in l-idonate metabolism have been given the designation idn (see Table Table11 for gene nomenclature).

TABLE 1

Gene and enzyme nomenclaturea
Gene designation
Gene product% Identity of proteinb
PreviousNew (acces- sion no.)
gntVidnK (P39208)d-Gluconate kinase45 (GntKc)
yjgVidnD (P39346)l-Idonate 5-dehydrogenase30.6 (sheep DHSOd)
yjgUidnO (P39345)5-Keto-d-gluconate 5-reductase56 (GNOe)
gntWidnT (P39344)l-Idonate transporter61 (GntTf)
yjgSidnR (P39343)l-Idonate regulator46 (GntRg)
Open in a separate windowaAll accession numbers are Swiss-Prot database accession numbers. bPercent identity of the amino acid sequence of the Idn protein to that of the protein shown in parentheses. cE. coli gluconate kinase encoded by gntK (P46859). dSheep sorbitol dehydrogenase encoded by sorD (P07846). eG. oxydans gluconate:NADP 5-oxidoreductase encoded by gno (P50199). fE. coli gluconate transporter encoded by gntT (P39835). gE. coli gluconate regulator encoded by gntR (P46860). (Part of this work has been presented previously [3].)  相似文献   

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