Part Two and Three The Applications of Gene Cloning and DNA Analysis in Research and Biotechnology Chapter 10. Studying gene location and structure Chapter 11. Studying gene expression and function Chapter 12. Studying genomics and post genomics Chapter 13. Production of protein from cloned genes Chapter 14 . Gene cloning and DNA analysis in medicines Chapter 15 . Gene cloning and DNA analysis in agriculture Chapter 16 . Gene cloning and DNA analysis in forensic science

Studying gene expression Chapter 11 Studying gene expression And function Studying the RNA transcript of a gene Studying the regulation of gene expression Identifying and studying the translation product of a cloned gene ●

The process of gene expression Figure 11.1

exons Figure 11.1

5’ -UTR 3’ -UTR Figure 11.1

1. Northern hybridization 11.1 Studying the RNA transcript of a gene Detecting the presence of transcripts of a gene and determining its nucleotide sequence 1. Northern hybridization

Northern hybridization If RNA from different tissues is run in different lanes of the gel, then the possibility that the gene is differentially expressed can be examined. R S L 4 8 12 16 20 24 28 32 36 40 44 48h Darkness Northern hybridization

Comparison between the sequence of the cDNA and the sequence of its gene will reveal the positions of introns and possibly the start and end points of the transcript. However, The cDNA synthesis might not continue all the way to the 5′end of the transcript, especially if the RNA is more than a few hundred nucleotides in length.

2. Transcript mapping by hybridization between gene and RNA The size can be measured by gel electrophoresis.

Locating a transcription start point by S1 nuclease mapping Figure 11.5

3. Transcript analysis by primer extension Figure 11.6

4. Transcript analysis by PCR Rapid Amplification of cDNA Ends (RACE)

11.1 Studying the transcript of a cloned gene 11.2 Studying the regulation of gene expression 11.3 Identifying and studying the translation product of a cloned gene R S L 4 8 12 16 20 24 28 32 36 40 44 48h Darkness

11.2 Studying the regulation of gene expression Figure 11.8 Possible positions for control sequences in the region upstream of a gene. The gene is switched on or off by the attachment of regulatory proteins to the control sequences Positive role or negative role Firstly, how to obtain the upstream of a gene?

11.2.1 The methods of cloning the promoter of a gene Using PCR Adapter PCR (接头PCR法) Reverse PCR (反向PCR法) Tail PCR (热不对称交错PCR)

◆ 接头PCR法

◆ 反向PCR法 Genomic DNA Known sequence analysis Genomic DNA digestion Ligation PCR Cloned cDNA sequence Upstream sequence Downstream sequence Genomic DNA RE Pu Pd RE Pd Pu Pd RE Pu

◆ Tail-PCR法 (Termal Asymmetric Interlaced PCR)又叫热不对称交错PCR Genomic walking

11.2.2 To determine the possible positions for control sequences in the upstream of a gene

1. Identifying protein-binding sites on a DNA molecule (a) Gel retardation of DNA-protein complex (凝胶阻滞检测） (b) Footprinting with DNase I (c) Modification interference assays 2. Identifying control sequences by deletion analysis (a) Reporter genes (b) Carrying out a deletion analysis

(a) Gel retardation of DNA-protein complex (凝胶阻滞检测） A bound protein decreases the mobility of a DNA fragment during gel electrophoresis.

Carrying out a gel retardation experiment Fig 11.10 Carrying out a gel retardation experiment ① Extraction of gDNA ② ③ ④

3 3 4 4 1 1 5 5 2 2

11.2.2.1 Identifying protein-binding sites on a DNA molecule (a) Gel retardation of DNA-protein complex (凝胶阻滞检测） (b) Footprinting with DNase I (c) Modification interference assays 11.2.2.2 Identifying control sequences by deletion analysis (a) Reporter genes (b) Carrying out a deletion analysis

(b) Footprinting (足迹法） Fig. 11.11 A bound protein protects a region of a DNA from degradation by a nuclease such as DNase I

Footprinting (足迹法） ① ② ③ ④

④

11.2.2.1 Identifying protein-binding sites on a DNA molecule (a) Gel retardation of DNA-protein complex (凝胶阻滞检测） (b) Footprinting with DNase I (c) Modification interference assays 11.2.2.2 Identifying control sequences by deletion analysis (a) Reporter genes (b) Carrying out a deletion analysis

(c) Modification interference assays

11.2.2.1 Identifying protein-binding sites on a DNA molecule (a) Gel retardation of DNA-protein complex (凝胶阻滞检测） (b) Footprinting with DNase I (c) Modification interference assays 11.2.2.2 Identifying control sequences by deletion analysis (a) Reporter genes (b) Carrying out a deletion analysis

Reporter genes 半乳糖苷酶，新霉素磷酸转移酶，氯霉素乙酰转移酶，二氢叶酸还原酶，潮霉素磷酸转移酶，荧光素酶，葡糖醛酸酶

Deletion analysis by restriction enzyme Fig 11.17 Deletion analysis. A reporter gene has been attached to the upstream region of a seed-specific gene from a plant.

Deletion analysis by PCR -232 ~ -1 -617 ~ -1 -1078 ~ -1 GUS GhNP1 GhNP3 GhNP2 GhNP1 GhNP2

11.1 Studying the transcript of a cloned gene 11.2 Studying the regulation of gene expression 11.3 Identifying and studying the translation product of a cloned gene 11.3.1 HRT (Hybrid-release translation) and HART (hybrid-arrest translation) can identify the translation product of a cloned gene 11.3.2 Analysis of proteins by in vitro mutagenesis

Cell-free translationsystems are cell extracts, usually prepared from germinating wheat seeds or from rabbit reticulocyte cells and containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis. AA Fig 11.18

① Hybrid-release translation Fig 11.19

② Hybrid-arrest translation Fig 11.20

③ We can also use the pGEM3Z vector to synthesize RNA in vitro Use these proteins, we can do some experiments, such as pull down, co-immunoprecipitation, etc, to uncover the mechanism of a gene product.

11.1 Studying the transcript of a cloned gene 11.2 Studying the regulation of gene expression 11.3 Identifying and studying the translation product of a cloned gene 11.3.1 HRT (Hybrid-release translation) and HART (hybrid-arrest translation) can identify the translation product of a cloned gene 11.3.2 Analysis of proteins by in vitro mutagenesis

Using mutation in the coding region to determine protein function Fig 11.21

Different types of in vitro mutagenesis techniques Fig 11.22

Arabidopsis T-DNA insertional mutants Different types of in vitro mutagenesis techniques Fig 11.24 Arabidopsis T-DNA insertional mutants

Oligonucleotide-directed point mutagenesis Fig 11.23 Protein function research important amino acid

Creating a point mutation in a cloned gene Fig 11.25 One method of using PCR to create a directed mutation

The end of chapter 11