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Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. IV. The product of the applied technologies

100%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

The Polymerase Chain Reaction and other molecular technologies were applied to isolate the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene. This research object was realized and the importance of this result has been discussed in view of the mechanisms of the regulation of gene expression.

2

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. II. The technology for the preparation of the template

88%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

The technology for the preparation of the template in the research aimed to isolate the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene is presented i.e. the linearisation of the template by Bam H I digestion, the purification of the template and the estimation of the template concentration.

3

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. IV. The product of the applied technologies

75%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

156-164

4

Cloning and expression of the two DNA fragments of the I-18 C region of Chironomus tentans. II. The effects of the applied technology

75%

Borowicz B. P.

Journal of Plant Protection Research

|

1998

|

tom 38

|

nr 1

The chromosomal I-18 C region of Chironomus tentans contains the I-18 C gene. Two different open reading frames (i.e. the ORF I and II) of two different transcripts (i.e. the 1.8 kb and 4.6 kb RNA) of the I-18 C gene of Chironomus tentans were isolated, at the DNA level, by the Polymerase Chain Reaction, then were cloned into the bluescript vector and finally were cloned into the pET-3a vector in order to translate them in T7 RNA polymerase / promoter system of E. coli. It was possible to obtain the ORF I overexpressed. In a case of the ORF II the low molecular weight polypeptide was detected, however it was not overexpressed, but still this polypeptide was strongly visible on the SDS-polyacrylamide gel.

5

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. II. Preparation of the intermediate vector - bluescript plasmid for ligation with the inserts

75%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments of the I-18 C gene in regard to the preparation of the intermediate vector i.e. the bluescript for the ligation with the ORF I and II reflecting DNA fragments, is presented. The main steps include the Eco RV digestion of the bluescript plasmid, the phenol / methylene chloride extraction of the plasmid, dephosphorylation of the bluescript plasmid and finally its extraction with the phenol and ethanol precipitation.

6

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. V. Different mechanisms of regulation regarding the open reading frames

75%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

Different mechanisms of regulation regarding the Open Reading Frames (ORFs) have been discussed to have a broader perspective that is necessary to evaluate the role of the ORFs of the I-18 C gene. This consideration includes the ORF II of the I-18 C gene which is the object of the presented research.

7

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. III. The DNA amplification technology

75%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

The technology used to amplify and isolate the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene of Chironomus tentans is described i.e. the Polymerase Chain Reactioin and the agarose gel electrophoresis of this reaction product, as well as the blunting reaction of the product and its isolation.

8

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. V. Different mechanisms of regulation regarding the open reading frames

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

165-171

9

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. II. The technological for the preparation of the template

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

145-148

10

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. I. The technology for the preparation of the primers

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

138-144

11

Isolation of the DNA fragment reflecting the open reading frame I of I-18 C gene of Chironomus tentans by the polymerase chain reaction. I.The technology for the preparation of the samples

63%

Borowicz B. P.

Prace Naukowe Instytutu Ochrony Roślin

|

1995

|

tom 36

|

nr 1-2

69-76

12

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. III.The DNA amplication technology

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

149-155

13

Isolation of the DNA fragment reflecting the open reading frame I of the I-18 C gene of chironomus tentans by the polymerase chain reaction. III The effect of the applied technologies

63%

Borowicz B. P.

Prace Naukowe Instytutu Ochrony Roślin

|

1995

|

tom 36

|

nr 1-2

83-93

14

Cloning and expression of two DNA fregments of the I-18 C region of Chironomus tentans. I. The scheme of the performed experiments and the applied technology

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1998

|

tom 38

|

nr 1

The I-18 C region of the polytenic chromosome of Chironomus tentans contains the I-18 C gene. Two DNA fragments, reflecting two different open reading frames (i.e.ORF I and II) of two different transcripts (i.e. 1.8 and 4.6 kb RNA) of the I-18 C gene were isolated, then were cloned into the intermediate vector and next to the final vector in order to translate them in T7 RNA polymerase / promoter system. The translation of the ORF I and II was performed to obtain the polypeptides of these ORFs for further study. Here, the scheme of the performed experiments and the applied technology that were necessary to realize the goal of this research, are presented.

15

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. III. Preparation of the inserts for ligation with the bluescript and the modification of the vector

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology applied for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments of the I-18 C gene of Chironomus tentans in regard to the preparation of the inserts for ligation with the bluescript and the modification of this intermediate vector, is described. The ORF I reflecting DNA fragment prepared for ligation with the bluescript vector, had one end blunt (i.e. the 5’ end with the Nde I restriction site) and the second end was sticky (i.e. the 3’ end after the Bam HI digestion of this insert). Subsequently, the bluescript vector for this ligation had also one end blunt (i.e. the 5’ end with the Nde I restriction site) and the second end was sticky (i.e. the 3’ end after the Bam HI digestion). The ORF II reflecting DNA fragment prepared for the ligation had both ends blunt and so the vector.

16

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. I. The technology for the preparation of the primers

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1997

|

tom 37

|

nr 1-2

The above mentioned technology in a case of the DNA fragment reflecting the Open Reading Frame II of the I-18 C gene is described i.e. the primers design, purification and phosphorylation of the oligonucleotide primers.

17

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IV. The ligation of the bluescript vector with the inserts

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments of the I-18 C gene in regard to the ligation reactions of the bluescript vector with the inserts, is presented. The main steps include: the calculation of the molar ratio of the bluescript plasmid to the ORF I and II reflecting DNA fragments as the inserts, the ligation reaction, the transformation of the E. coli cells of the XL- 1 strain with the ligation reactions products and growing of the transformed bacterial cells.

18

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. V. Identification of the bacterial colonies, containing the recombinant bluescript plasmids

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology applied for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the identification of the bacterial colonies, of E. coli XL-1 strain, containing the recombinant bluescript plasmids, is described. The particular steps include: the α-complementation test, growing of the preliminary selected bacterial colonies in culture, isolation of the plasmids from these colonies, the Nde I and Bam HI digestion of the plamids mini-preparations, analysis of the digestion products and the identification of the bacterial colonies, containing the bluescript plasmids with the cloned inserts by the agarose gel electrophoresis (2% gel, 1 x TBE buffer).

19

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VII. The preparation of the inserts and the translational vector - pET-3a for ligation

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology applied for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the preparation of the inserts and the translational vector pET-3a for ligation, is described. The main steps of the preparation of the inserts include: digestion of the isolated recombinant bluescript plasmids, carrying the inserts, with the Nde I and Bam HI, purification and isolation of the inserts after the digestion, by the preparative agarose gel electrophoresis (2% gel, 1 x TAE buffer) and then by the centrifugation through the filtration device of the isolated DNA fragment from the gel and also estimation of the inserts concentration by the agarose gel electrophoresis (2% gel, 1 x TBE buffer). The main steps of the preparation of the pET-3a vector included: transformation of the competent E. coli cells of the XL-1 strain with the pET-3a, growing of the transformed bacteria on agar plates (with ampicillin) and then growing of the isolated transformed bacterial colonies in culture to finally isolate the plasmid, digestion of the plasmid with the Nde I and Bam HI, purification of the isolatcd plasmid, after the digestion, by the preparative agarose gel electrophoresis (0.8% gel, 1 x TAE buffer), centrifugation through the filtration device of the isolated DNA fragment from the gel, phenol extraction of the isolated plasmid, estimation of the concentration of the plasmid by the agarose gel electrophoresis (0.8% gel, 1 x TBE buffer).

20

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VI. The DNA sequencing of the cloned inserts

63%

Borowicz B. P.

Journal of Plant Protection Research

|

1999

|

tom 39

|

nr 1

The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the DNA sequencing of the cloned inserts, is presented. The object of the DNA sequencing of the cloned inserts, was to finally confirm and prove the sequence of the inserts as the sequence of the ORF I and II reflecting DNA fragments of the I-18 C gene of Chironomus tentans.

Ograniczanie wyników

21 Journal of Plant Protection Research

3 Prace Naukowe Instytutu Ochrony Roślin

1 Journal of Applied Genetics

25 Borowicz B. P.

1 2005

9 1999

2 1998

10 1997

3 1995

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. IV. The product of the applied technologies

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. II. The technology for the preparation of the template

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. IV. The product of the applied technologies

Cloning and expression of the two DNA fragments of the I-18 C region of Chironomus tentans. II. The effects of the applied technology

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. II. Preparation of the intermediate vector - bluescript plasmid for ligation with the inserts

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. V. Different mechanisms of regulation regarding the open reading frames

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. III. The DNA amplification technology

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. V. Different mechanisms of regulation regarding the open reading frames

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. II. The technological for the preparation of the template

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. I. The technology for the preparation of the primers

Isolation of the DNA fragment reflecting the open reading frame I of I-18 C gene of Chironomus tentans by the polymerase chain reaction. I.The technology for the preparation of the samples

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaktion. III.The DNA amplication technology

Isolation of the DNA fragment reflecting the open reading frame I of the I-18 C gene of chironomus tentans by the polymerase chain reaction. III The effect of the applied technologies

Cloning and expression of two DNA fregments of the I-18 C region of Chironomus tentans. I. The scheme of the performed experiments and the applied technology

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. III. Preparation of the inserts for ligation with the bluescript and the modification of the vector

Isolation of the DNA fragment reflecting the open reading frame II of the I-18 C gene of Chironomus tentans by the polymerase chain reaction. I. The technology for the preparation of the primers

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. IV. The ligation of the bluescript vector with the inserts

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. V. Identification of the bacterial colonies, containing the recombinant bluescript plasmids

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VII. The preparation of the inserts and the translational vector - pET-3a for ligation

Cloning of the DNA fragments reflecting the open reading frame I and II of the I-18 C gene of Chironomus tentans. VI. The DNA sequencing of the cloned inserts