共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
4.
In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup+ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap+ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product. 相似文献
5.
Yasunobu Kano Katsuaki Osato Morimasa Wada Fumio Imamoto 《Molecular & general genetics : MGG》1987,209(2):408-410
Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene. 相似文献
6.
A Bacillus cellulase gene coding for carboxymethylcellulase (CMCase) has been cloned in Escherichia coli using pBR 322 as a vector. The gene was expressed independently of its orientation in the cloning vector showing enzyme activity 40 times greater than that produced by the original Bacillus species. The high production of CMCase in E. coli by the foreign gene did not impede growth of the host cells and the E. coli produced CMCase responded to various pH values and temperatures in the same way as that produced by the gene donor cells. 相似文献
7.
A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein. 相似文献
8.
A DNA fragment containing both the Escherichia coli
-xylose isomerase (
-xylose ketol-isomerase, EC 5.3.1.5) gene and the
-xylulokinase (ATP:
-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The
-xylose isomerase gene was separated from the
-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The
-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the
-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first
-xylose isomerase gene that has been isolated and extensively purified from any organism.
-Xylose isomerase, the enzyme product of the
-xylose isomerase gene, is responsible for the conversion of
-xylose to
-xylulose, as well as
-glucose to
-fructose. It is widely believed that yeast cannot ferment
-xylose to ethanol primarily because of the lack of
-xylose isomerase in yeast.
-Xylose isomerase (also known as
-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the
-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting
-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 相似文献
9.
The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4 kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate. 相似文献
10.
The Schizosaccharomyces pombe maltase structural gene (SPMAL1+) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg2+, Ca2+, Na+, and Ba2+, but was strongly inhibited by Hg2+, Ag+ and Cu2+, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools. 相似文献
11.
12.
Carol A. Miles Andrew Mountain G. R. K. Sastry 《Molecular & general genetics : MGG》1987,206(1):169-173
Summary The trpE gene of Agrobacterium tumefaciens C58 was cloned from a gene library by complementation in Escherichia coli. It was shown to be unlinked to trpD gene in this organism. It was also shown that the nontumorigenic phenotype of tryptophan auxotrophs of A. tumefaciens could be complemented by addition of exogenous tryptophan. The role of bacterially synthesised tryptophan in the process of tumour formation is discussed.Abbreviations Ap
ampicillin
- Cm
chloramphenicol
- Gent
gentamycin
- Km
kanamycin
- dATP
deoxyadenosine 5-triphosphate
- IAA
indole acetic acid
- NB
nutrient broth
- MinAB
minimal Agrobacterium medium 相似文献
13.
次级淋巴组织趋化因子(SLC)基因克隆及在大肠杆菌中的表达 总被引:1,自引:0,他引:1
次级淋巴组织趋化因子(SLC)是通过搜索表达序列标签(EST)数据库克隆出来的一CC类趋化因子。以人SLC序列为蓝本,利用重叠PCR(SOE-PCR)的方法获得了适宜在大肠杆菌中表达的SLC基因,将此序列分别克隆至表达载体pTMF和pALM中,转化大肠杆菌,诱导表达。Western Blotting鉴定结果表明目的蛋白以可溶蛋白和包涵体两种形式表达,两种形式的蛋白所占比例依培养和诱导条件的不同而变化。对两种形式的表达产物分别用Ni-NTA金属亲和层析和包涵体复性方法纯化在实验中还对纯化条件进行了探索。对纯化蛋白的电泳结果显示:纯化样品的分子量比预期的分子量要大。 相似文献
14.
Paul G. Debenham Michael B. T. Webb John Law 《Molecular & general genetics : MGG》1988,215(1):156-160
Summary A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones. Iso-electric focusing of this gene product suggests it may function in a complex. 相似文献
15.
Ma Ting Li Shanshan Li Guoqiang Wang Renjing Liang Fenglai Liu Rulin 《Frontiers of Biology in China》2006,1(4):375-380
Dibenzothiophene (DBT) monooxygenase (DszC) catalysis, the first and also the key step in the microbial DBT desulfurization,
is the conversion of DBT to DBT sulfone (DBTO2). In this study, dszC of a DBT-desulfurizing bacterium Rhodococcus sp. DS-3 was cloned by PCR. The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank. The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E. coli BL21 strain. The expression amount of DszC was about 20% of total supernatant at low temperature. The soluble DszC in the
supernatant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.
Only one band was detected by Western-blotting, which is for the antibody released in mouse against purified DszC in the expression
product of BL21 (DE3, paC5) and Rhodococcus sp. DS-3. The activity of purified DszC was 0.36 U. DszC can utilize the organic compound such as DBT and methyl-DBT, but
not DBT derivates such as DBF, which has no sulfur or inorganic sulfur.
__________
Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(6): 1–6 [译自: 南开大学学报 (自然科学版), 2005, 38(6): 1–6] 相似文献
16.
Summary From the specialized transducing bacteriophage cysB, recombinant phages cysB242 and cysB257 have been obtained, each of which carries an amber mutation in the cysB cistron. A comparison of polyacrylamide gel electrophoretic profiles of labelled extracts from uv-irradiated bacteria that had been infected with cysB
+ or with cysB-amber phages, led to the identification of a 39,000-dalton polypeptide as the product of the cysB gene. The native protein was purified to near radiochemical purity and was found to be an oligomer with an isoelectric point close to pH 7. 相似文献
17.
Renkichi Takata Tsunehiro Mukai Michiko Aoyagi Katsuji Hori 《Molecular & general genetics : MGG》1984,197(2):225-229
Summary The nucleotide sequence of the ribosomal protein gene rpsO (S15) and its flanking region were determined. The amino acid sequence of S15 protein deduced from the nucleotide sequence is in good agreement with the published amino acid sequence with one exception. The nucleotide sequence shows two probable promoter sites about 100 nucleotides upstream from the initiation codon (AUG) of rpsO. Inspection of the sequence also revealed structural homology between the distal part of rpsO and the reported S15 binding region in 16S rRNA. 相似文献
18.
19.
Tatsushi Mogi Hiroshi Yamamoto Toshifumi Nakao Ichiro Yamato Yasuhiro Anraku 《Molecular & general genetics : MGG》1986,202(1):35-41
Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations
pCMB
p-chloromercuribenzoate
- DM
Davis and Mingioli
- Ap
ampicillin
- NTG
N-methyl-N-nitro-N-nitrosoguanidine
- EMS
ethylmethane sulfonate
- Str
streptomycin
- Tet
tetracycline
- Ac
l-azetidine-2-carboxylic acid
- DHP
3, 4-dehydro-d,l-proline
- MTT
3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide
- Tris
tris(hydroxymethyl)aminomethane
- EDTA
ethylenediamine tetraacetic acid
- Kan
kanamycin
- Spc
spectinomycin 相似文献