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Metody i techniki biologii molekularnej w biotechnologii środowiskowej

100%

Raszka A. , Ziembińska A. , Wiechetek A.

Czasopismo Techniczne. Środowisko

2009

tom R. 106, z. 2-Ś

101-114

W ostatnich latach pojawiło się dużo różnorodnych metod pozwalających na analizę składu biocenozy oraz rozmieszczenia przestrzennego mikroorganizmów. Metody molekularne górują nad tradycyjnymi technikami, gdyż nie są uzależnione od hodowli mikroorganizmów na podłożach mikrobiologicznych. Jest to ważna cecha metod analitycznych, ponieważ przyspiesza to całą procedurę badawczą. Dodatkowo metody oparte na analizie materiału genetycznego charakteryzują się dużą czułością i powtarzalnością. W artykule opisano metody najczęściej wykorzystywane w biotechnologii środowiskowej.

During last several years a wide variety of methods useful in the analyzing of biocenosis' composition and spatial microorganisms distribution appeared. A significant aspect of molecular techniques usage is its independence from traditional microbiological methods. It is an important analytical feature, because it enables a speedier research. Additionally, methods based on genetic material are more sensitive and repeatable. This article contains the description of the techniques of the most commonly used in environmental biotechnology.

Isolation, cloning and expression of DNA fragments reflecting open reading frames of 1.8 and 4.6 kb RNAs of the I-18 C gene of Chironomus tentans

100%

Borowicz B. P.

tom 46

nr 1

This study, based on the genetic engineering approach, concerns the regulation of the insect gene expression, but it has also a strong significance for plant protection, which is associated with the future possibility to control insect development more effectively, especially in those species that are pests of cultivated plants. The I-18 C gene of Chironomus tentans is activated by heat-shock and the steroid hormone - ecdysone. This gene produces different transcripts (at least four) by alternative splicing. Two different open reading frames, i.e. ORFs I and II of the two main transcripts of this gene, i.e. the 1.8 and 4.6 kb RNA, had been isolated, at the DNA level, as the ORFs reflecting DNA fragments, using the polymerase chain reaction, then were cloned into the bluescript vector and finally were cloned into the pET-3a vector to be translated in the T7 RNA polymerase/promoter system of E. coli. As the preliminary outcome of these experiments it was possible to obtain ORF I overexpressed as the polypeptide, whereas ORF II was expressed as the polypeptide that was strongly visible on SDS-polyacrylamide gel. This could facilitate further studies regarding the mechanisms of expression of this gene, including the level of expression of this gene during the C. tentans development.

Isolation and preliminary sequence characterization of beta-amylase gene promoters in rye [Secale cereale L.]

100%

Sadowski J. , Rorat T. , Cooke R. , Delseny M.

Journal of Applied Genetics

1997

tom 38

nr 3

The isolation of rye ß-Amy1 and ß-Amy2 gene promoters from nuclear DNA using the inverse polymerase chain reaction (IPCR) technique and characterization of their sequences are presented. The conservation of ß-amylasc coding sequences allowed for simultaneous IPCR amplification of two different promoters with primers designed on the basis of the single known cDNA sequence. Two ß-amylasc gene promoters display a low sequence similarity (47%). Beside consensus TATA and CCAAT boxes, other sequence motives common to both promoters were found. In addition, the homology of amino acid sequences of plant ß-amylases available in the database is discussed.