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High temp ersistant triphosphate guanosine cyclohydrolase gene sequence and coded polypeptide and method for preparing said polypeptide

A guanosine triphosphate, high temperature resistant technology, applied in the field of mutation or genetic engineering, can solve problems such as unknown details

Inactive Publication Date: 2002-10-02
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the reaction mechanism of guanosine triphosphate cyclohydrolase has been proposed in biochemical and crystallographic studies, the details of this complex reaction sequence are still unknown

Method used

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  • High temp ersistant triphosphate guanosine cyclohydrolase gene sequence and coded polypeptide and method for preparing said polypeptide
  • High temp ersistant triphosphate guanosine cyclohydrolase gene sequence and coded polypeptide and method for preparing said polypeptide
  • High temp ersistant triphosphate guanosine cyclohydrolase gene sequence and coded polypeptide and method for preparing said polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Construction of a sequencing library

[0034] The sequencing library was constructed using the genome-wide shotgun method. Firstly, Tengchong thermophilic anaerobic bacteria were cultured according to (Yanfen Xue, 2000) modified MB medium (Balch et al., 1979), bacteria were collected according to Marmur (1961) method, and total DNA was extracted. In order to ensure the randomness of sequencing library construction and avoid the problem of hot spots of breakage to the greatest extent, a variety of methods and principles of library construction under different conditions were adopted. Firstly, physical shearing method (including ultrasonic method and shearing with Hydroshear Machine) was used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme diges...

Embodiment 2

[0035] Example 2: Genome Sequencing

[0036] When sequencing the genome of thermophilic anaerobic bacteria in Tengchong, two automatic sequencers were mainly used: A BI377 and MegaBACE 1000. These two sequencers use the principle of electrophoresis for sequencing (see Figure 2), and can complete 96 samples each time. ABI377 is a product of PE Company, which is a kind of ABI series. It belongs to the slab gel electrophoresis sequencer. MegaBACE 1000 is a product of Pharmacia, which belongs to capillary gel electrophoresis sequencer.

Embodiment 3

[0037] Example 3: Basecalling and sequencing quality monitoring

[0038] The so-called basecalling refers to the process of obtaining the correct base sequence from the raw data file obtained on the sequencer. Since the sequencer obtains the intensity change traces (traces) of light of different wavelengths corresponding to the four bases A, T, G, and C, it is necessary to use a computer to adopt a certain algorithm to correctly identify the bases corresponding to the different traces. . We used Phred software (Ewing B, Hillier L, 1998) because its results are more reliable and its output is more convenient for further analysis by other programs in the same software package.

[0039] The algorithm principle of Phred's basecalling is to judge the base type based on the shape, distance, and signal-to-noise ratio of each peak in the trajectory, and at the same time give the credibility information for this base, that is, the sequencing quality of the base. In large-scale sequen...

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Abstract

The present invention discloses a high-temp. resistant triphosphoric acid guanosine cyclohydrolase gene, its coded polypeptide and preparation method. It relates to coding the separate DNA with activity or its function-identical variant and producing the polypeptide with high-temp. resistant triphosphoric acid guanosine cyclohydrolase activity or its function-identical variant by DNA recombination technology and said separate DNA. Said ivnention uses the sequencing and analysis of Tengchong thermophilic anaerobe hologenome as basis to clone and separate the high-temp. resistant triphosphoric acid guanosine cyclohydrolase gene, and utilizes said gene to prepare its transgenic microbe or transgenic animal and plant for producing said high-temp. resistant triphosphoric acid guanosine cyclohydrolase and recover the enzyme obtaining said gene code.

Description

technical field [0001] The present invention relates to mutation or genetic engineering, in particular to a high-temperature-resistant guanosine triphosphate cyclic hydrolase gene sequence, encoded polypeptide and a preparation method. Background technique [0002] GTP (guanosine triphosphate) cyclohydrolase (GTP-CH-I; E.C.3.5.4.16), which catalyzes the conversion of GTP into dihydroneopterin triphosphate, is one of the reactions in the biosynthesis of tetrahydrobiopterin Rate-limiting enzyme whose activity is sensitive to feedback proteins inhibited by tetrahydrobiopterin. In mammals, dihydroneopterin triphosphate can be converted into tetrahydrobiopterin by a series of actions of 6-acetone tetrahydrofolate synthesis and mepterin reductase. [0003] Tetrahydrobiopterin acts as an important cofactor for nitric oxide synthesis, glycerol ether monooxygenase, and pterin-demanding monooxygenase. Tetrahydrobiopterin is a cofactor for tyrosine hydroxylase,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/78C12N15/55C12N15/63C12Q1/68
Inventor 包其郁杨焕明张立敏汪建王光信
Owner HUADA GENE RES & DEV CENT HANGZHOU
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