Chapter thirteen** Gene expression regulation

Section 1** Basic concept, specificity and types of gene expression Section 2** Basic principle of gene expression regulation Section 3** regulation of gene expression in prokaryote Section 4** regulation of gene expression in eukaryote

Section 1 The basic concept, specificity and types of gene expression

1.1 The concept of gene expression = + transcription translation

Interaction among large biomolecules 1 proteins 2 RNAs 1 proteins 2 RNAs DNAs RNAs Proteins Interaction among large biomolecules

1.2 The specificity of gene expression in the time and the space

1.2.1 The temporal specificity of gene expression The temporal specificity is that some specific genes in genome are expressed in order of specific time The temporal specificity is also stage specifi-city in the polycellular biosomes The expressed genes in early developmental steps are more than in other steps in polycellular biosomes The expressed genes relate with biological function.

1.2.2 The spatial specificity of gene expression In the polycellular biosomes the spatial speci- ficity of gene expression is that one or some specific genes in the genome are expressed in different systems, organs, tissues and cells in order of space. The spatial specificity of gene expression is also known as tissue specificity or cell specificity. The expressed genes relate with biological function.

1.3 Types of gene expression 1.3.1 Constitutive gene expression 1.3.2 Inductive and repressive gene expression

1.3.1 constitutive gene expression  Some genes in genome are known as housekeeping genes.  The expression of housekeeping genes in genome are also called constitutive gene expression.  Constitutive gene expression is continual in most cells.

less effected by environment factors.  The expressive products of constitutive genes are absolutely necessary in all over life process.  The expression of constitutive genes are less effected by environment factors.  The constitutive gene expression are only effected by interacting between promoter and RNA polymerase.

1.3.2 Inductive and repressive gene expression  The expression of some genes in genome are more effected by environment factors.  The increase of gene expression which is effected by environment factors are called induction, in contrast, the decrease of that are repression.

genes are regulated by other factors,  The expression of induced or repressed genes are regulated by other factors, besides interaction between promoter and RNA polymerase.  The special elements are located in the regulation region of induced or repressed genes.  Induction and repression of gene expression correspond with each other.

1.4 Biological significance of gene expression regulation  acclimation  keep growth and proliferation  keep individual development and differentiation

Section 2 Basic principle of gene expression regulation

2.1 multilevel regulation of gene expression check point activity of gene structure in genome DNA amplification DNA rearangement DNA methylation  initiation of RNA transcription*  process of RNA post-transcription  transport of RNA post-process  initiation of protein translation  process of protein post-translation

2.2 basic factors of gene expression regulation regulation protein RNA polymerase specific DNA sequence

2.2.1 specific DNA sequence 2.2.1.1 promoter of prokaryotic genes transcriptional initiation site 2.2.1.1 promoter of prokaryotic genes “TTGACA” spacer “TATAAT” spacer +1 N17 N7 trp TTGACA TTAACT A N16 N7 tRNATyr TTTACA TATGAT A N17 N6 TTTACA TATGTT A lac N16 N7 recA TTGATA TATAAT A N18 N6 ara BAC CTGACG TACTGT A -35 region -10 region consensus sequence

2.2.1.2 the prokaryotic operon in 1961 by Jacob and Monod.  The concept of the operon was first proposed in 1961 by Jacob and Monod.  An operon is a whole unit of prokaryotic gene expression which includes a set of structural genes and its promoter, operator and other control elements which are recognized and bond by regulatory gene products.

Structural genes region Regulatory region Inhibitor gene Gene 1 Gene 2 Gene 3 Structural genes region O p 3’ 5’ i gene region RNA polymerase repressor  The operator is a site bond with the repressor.  The operator mediates a negative regulation of operon  The operator is next to the promoter.  The operator sites in downstream of the promoter.  The operator overlaps partly with the promoter some time.

2.2.1.3 Other regulator of prokaryotic operons  special DNA sequence in some prokaryotic operons  can bind with activator of RNA polymerase  increase transcription of operons  mediate positive regulation of operons  The positive regulation is not main mechanism about gene expression regulation of operons, but the negative regulation is .

2.2.1.4 cis-acting elements of eukaryotic gene  The cis-acting elements are DNA fragments.  The cis-acting elements are the regulator of eukaryotic gene transcription  There are cis-acting elements in the flakings or the introns of eukaryotic gene.  The cis-acting elements include promoter,enhancer, silencer and so on.

eukaryotic genome RNA polymerase gene coding region enhancer silencer promoter 3’ 5’ exon intron exon  The eukaryotic genes are monocistron.  there are not the operons structure in eukaryotic genome

2.2.2.1 regulation protein of prokaryotic genes  specific factors： decide identification and bind between RNA polymerase and specific promoter  repressor： bind operator and repress gene transcription  activator: bind a special DNA sequence next to promoter advance to bind between RNA polymerase and promoter and to form transcription initiation complex.

2.2.2.2 regulation protein of eukaryotic genes  transcription factor, it is also trans-acting factor.  cis-acting protein protein A trans-acting protein trans-acting factor trans-regulation PA A PB B protein B cis-acting protein cis-regulation

2.2.3.1 promoter of prokaryotes/eukaryotes effect on RNA polymerase  The promoter of prokaryotes/eukaryotes is consist of transcriptin initiation site, RNA polymerase identification and binding site and other regulation elements.  The promoter of eukaryotes is more complicated than that of prokaryotes.

polymerase effects directly on frequency of gene  The sequence of different promoter is certain different.  The affinity of prokaryotic promoter with RNA polymerase effects directly on frequency of gene transcription initiation  The affinity between eukaryotic RNA polymerase and promoter is less, when RNA polymerase is single.  The eukaryotic RNA polymerase can bind with promoter after forming complex with basic transcription factor.

2.2.3.2 regulation proteins effect on activity of RNA polymerase  Specific promoter decides basic transcription frequency of genes.  The regulation proteins can change transcription  The conformation or the expression level in the cell of regulation proteins gets a change under stimulation of environment signal.

interaction in transcription regulation of eukaryotic genes 2.2.4 DNA-protein and protein-protein interaction in transcription regulation of eukaryotic genes 2.2.4.1 DNA-protein interaction  identification and bind between cis-acting proteins or trans-acting factors and cis-acting elements  Its interaction is a non-covalent bond.  form DNA-protein complex finally

2.2.4.2 protein-protein interaction  The most regulation protein can form homodimer, heterodimer, homopolymer or heteropolymer before binding with cis-acting element.  ability of some regulation protein to bind with its cis- acting element is increased or decreased after polymerization.  Some regulation protein don’t bind with DNA, but can effect the activity binding between DNA and other regulation protein by protein-protein interaction.

Section 3 Regulation of prokaryotic gene expression 3.1 Characters of transcribed regulation in prokaryotic genes 3.2 Regulation of transcribed initiation in 3.3 Regulation of transcribed termination in 3.4 Regulation of proteic translation in prokaryote

3.1 characters of transcriptional regulation in prokaryotic genes 3.1.1 The function of  factors The  factors bind a special element in 5’ flaking region of genes in the stage of transcribed initiation The  factors decide the specificity of transcribed genes The  factors mediate holoenzyme of RNA polymerase binding to specific promoter of genes Different  factors decide transcription of different genes.

3.1.2 universality of operon model  the most prokaryotic genes  There are many operons in order in prokaryotic genome.  Don’t discover operon in eukaryotic genome. 3.1.3 universality of repression mechanism  The repression mechanism is the main mechanism of transcriptional regulation of prokaryotic genes

3.2 regulation of transcribed initiation in prokaryotic genes

structural genes region 3.2.1 stucture of lactose operon C regulatory region Inhibitor gene Gene Z Gene Y Gene A structural genes region O p 3’ 5’ i gene region 1000bp 100bp 3520bp 760bp 810bp base pair: peptide : (MW kDa) 37 repressor (4 polymer) 32 30 135 -galactosidase lactose cell galactose + acetyl CoA acetylgalactose glucose - galactoside transacetylase(2 polymer) lactose permease (2 polymer)

Primary structure of lac operon regulation region Catabolite gene activation protein site -10 site -35 site CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGAGCGGA TAACAATTTCACAC CTCATTAGG ACTCGATTGAGTGTAATTA 5’ 3’ Operon region 20bp RNA polymerase binding region or promoter region Primary structure of lac operon regulation region 5’ TATAAT 3’ Pribnow box

3.2.2 regulated mechanism of lac operon genes 3.2.2.1 repressor regulate negatively transcription of lac operon genes z y a i o p RNA polymerase repressor (4 polymer) z y a i o p repressor (4 polymer) RNA polymerase mRNA + galactose

+ regulate positively transcription of lac operon genes 3.2.2 catabolite gene activation protein(CAP) regulate positively transcription of lac operon genes CAP site -35 -10 1 5’ 3’ CAP cAMP RNA polymerase -35 -10 at glucose presents CAP cAMP + cAMP at glucose absents

+ + 3.2.3 correspond between negative regulation of repressor and positive regulation of CAP in gene transcription control of lac operon CAP site 5’ 3’ -35 -10 RNA polymerase inhibitor gene 3.2.3.1 glucose concentration is lower and lactose concentration is higher cAMP at glucose absents CAP + Repressor (4 polymer) + galactose

+ 3.2.3.2 glucose concentration is higher and lactose CAP site 5’ 3’ -35 -10 RNA polymerase inhibitor gene cAMP at glucose presents CAP + 3.2.3.2 glucose concentration is higher and lactose concentration is lower Repressor (4 polymer)

The negative regulation mechanism of the repressor cooperate with the positive regulation mechanism of the CAP control gene transcription of lac operon by kind and concentration of carbohydrate from the environment.

3.3 regulation of transcribed termination in prokaryotic genes

3.3.1 Dependent upon  factor Terminator Promoter Gene RNA polymerase 5’ c  new RNA 5’ c 

coding strand template strand 3.3.2 model about transcription termination for independent to  factor RNA polymerase coding strand 5’ template strand 5’ 5’ new RNA transcript

G A C C U G A U U U U-OH 3’ 5’ U 5’…GCCGCCAGUUCGGCUGGCGGCAUUUU…3’ RNA 5’…GCCGCCAGTTCGGCTGGCGGCATTTT… 3’ terminator The RNA made from the DNA palindrome is self-complementary and so formed a internal hairpin structure followed by a few U bases. The signals that terminates transcri-ption are localized in the gene 3’ end. A simple termination signal is a GC- rich region that is a palindrome, followed by AT-rich sequence. DNA

3.3.3 attenuation regulation mechanism of gene transcription of trp operon in E.coli L Trp operon R E D C B A p O attenuater Synthesis of tryptophan in E. coli O HO H2C COOH C Chorismic acid NH2 Anthranilic acid synthetase CH2 N CH OH P Indolglycerol phosphate Indoglycerol phosphate synthetase Tryptophan

The regulation mechanism of trp operon at a few tryptophans present or tryptophans absent L Inactive repressor R E D C B A p O whole mRNA at a lot of tryptophans present Trp + Repressor RNA polymerase or fragmentary mRNA

tryptophans absent ribosome RNA polymerase RNA DNA lot of tryptophans K A I F L G 1 2 3 4 ribosome Try code RNA polymerase RNA DNA W R tryptophans absent lot of tryptophans

3.4 regulation of proteic translation in prokaryote

3.4.1 autoregulation/autogenous control DNA started region mRNA 5’ 3’ same mRNA Protein

3.4.2 antisene control Antisens RNA DNA started region 5’ 3’ mRNA Protein mRNA DNA 5’ 3’ started region Antisens RNA

Section 4 Regulation of eukaryotic gene expression 4.1 character of eukaryotic genomic structure 4.2 character of expressional regulation of eukaryotic genes 4.3 regulation of transcription for RNA pol I and RNA pol III 4.4 regulation of transcriptional initiation for RNA pol II 4.5 regulation of transcriptional termination for 4.6 regulation of past-transcription for RNA 4.7 regulation of translation for protein

4.1 character of eukaryotic genomic structure  The eukaryotic genome is very great.  The structure of eukaryotic genome is very complex.  There is only a gene in a transcription unit of eukaryotic genome.  There are a lot of repeat sequences in eukaryotic genome.  gene is discontinuous, there are noncoding sequences in the most eukaryotic gene.

4.2 character of transcription regulation of eukaryotic genes 4.2.1 three RNA polymerases in eukaryote  RNA polymerase Ⅰ，45s-rRNA(28S, 18S, 5.8S) RNA polymerase Ⅱ*，hnRNA(mRNA), a part of snRNA  RNA polymerase Ⅲ，5s-rRNA, tRNA, a part of snRNA

 The every RNA polymerase is consist of about 10 subunits.  The some subunits are in common for every RNA polymerase , for example TATA box-binding protein (TBP).  The some subunits are special for a RNA polymerase.  TFⅡD is core of polymeraseⅡ.  TFⅡD is consist of TBP and TBP-related factor.

4.2.2 structural character of active gene region  There are hypersensitive sites of DNaseⅠin the flanking regions of active gene, near regulation protein binding sites. Gene coding region Promoter Silencer or Enhancer site

DNA RNA

activity of gene structure dsDNA supersolenoid chromatid chromosome solenoid chromatin nucleosomes transcription bubble RNA activity of gene structure 2 1 3 4 5 6

of transcription region DNA in front of RNA  When gene is activated, topology structure of transcription region DNA in front of RNA polymerase is positive superhelix conformation,that one in back of of RNA polymerase is negative superhelix conformation. RNA polymerase negative superhelix positive superhelix

DNA is propitious to form nucleosomes GCGCGCGC  The negative superhelix conformation of DNA is propitious to form nucleosomes again, positive one is propitious to separate histone in nucleosomes  The methylation of CpG sequence in active gene flaking region is lower. Gene

often as follows: from nucleosome. structure becomes instability.  The histons in active gene region change often as follows:  The rich-Lys H1-like histons are decreased.  The ability of DNA to form 30nm fasciculi is reduced  The instability of dimer H2A-H2B is increased.  The dimer H2A-H2B is prone to be replaced out from nucleosome.  The histons is prone to be modified, in result its structure becomes instability.  H3 histon sulfhedryl is exposed out.

4.2.3 The positive regulation is main in gene transcription regulation of eukaryotes  The most transcriptional regulation proteins are transcriptional activation proteins.  The affinity between RNA polymerase and pomoter in eukaryotes is very weaker or not.  RNA polymerase must depend on one or many activation proteins to bind with pomoter.  The positive regulation is universality in gene transcription regulation of eukaryotes.

a gene regulation region, in result that  The eukaryotic genome is very great.  There are many cis-acting elements in a gene regulation region, in result that specificity of interaction between activation protein and DNA is increased.  Many activation proteins regulate a gene, therefore the regulation efficiency is higher.  A activation proteins regulate many gene, therefore the regulation is more economical.

4.2.4 The transcription and the translation are separated in different area , this is prone to regulate exactly in gene transcription 4.2.5 The process of post-transcription modification is more complex and perfect, therefore links of gene expression regulation is increased.

4.3 regulation of transcription for RNA polⅠ and RNA pol Ⅲ

4.3.1 control of transcription for RNA polⅠ rDNA gene -45 +20 -156 -107 core element upstream control element rRNA RNA polⅠ upstream binding factor 1, UBF-1 selectivity factor 1, SL-1

4.3.2 control of transcription for RNA pol Ⅲ +1 TGGCNNAGTGG tDNA gene GGTTCGANNCC TGGCNNAGTGG GGTTCGANNCC TF Ⅲ C TF Ⅲ B RNA pol Ⅲ TGGCNNAGTGG GGTTCGANNCC TGGCNNAGTGG GGTTCGANNCC tRNA

+1 5s rDNA gene TF Ⅲ A TF Ⅲ B TF Ⅲ C RNA pol Ⅲ 5s rRNA

4.4 regulation of transcriptional initiation for RNA polⅡ

4.4.1 cis-acting element according to function, main including promoter, enhancer, silencer and so on

RNA polymerase exon intron exon silencer promoter gene coding region enhancer silencer promoter 3’ 5’ exon intron exon RNA polymerase

4.4.1.1 promoter of other functional elements which control  It is consist of RNA polimerase binding-site, a set of other functional elements which control transcription and a transcription initiation site at least.  The length of every functional elements in the promoter is about 7-20 bp.  Promoter sequence of different genes is a little different.  Most gene promoter sequence have TATA box.

functional element in the promoter.  TATA box is the most basic and important functional element in the promoter.  The consensus sequence of TATA box is TATAAAA locating in -25  -30 bp region of transcription start site upstream.  TATA box controls veracity and frequency of gene transcription.  GC box（GGGCGG）and CAAT box （GCCAAT）is more frequent in the promoter  GC box and CAAT box locate in -30  -110 bp region of transcription start site upstream.

region of the gene certainly.  The promoter locates in upstream of coding region of the gene certainly.  The promoter has strict direction.  The most simple promoter is consist of TATA box and transcription start site, but the typical promoter often contains also GC box and/or CAAT box in upstream of TATA box.

contains many transcription start sites,  The promoter of some genes don’t contain TATA box.  The promoter without TATA box always contains many transcription start sites, and contains rich-GC sometimes.  The gene that contains the promoter without TATA box is keeping housegene or the gene which plays a role in fetation, tissue differentiation, tissue damage regeneration and so on.

4.4.1.2 enhancer transcription and decides space-time specificity of  The enhancer is a DNA fragment.  The enhancer increases activation of gene transcription and decides space-time specificity of gene transcription. gene enhancer 5’ 3’ 72bp core sequence TGTGGAATTAG

 The role of the enhancer don’t relate with its distance from transcription start site. The enhancer is very far from gene transcription start site(1~30/50kb) and locates not only in genic upstream, but also in genic downstream, sometimes in the intron.  Some important functional elements，for example core DNA sequence which is bond with special transcription factors, is in the enhancer and the promoter at one time. Gene regulatory proteins Gene regulatory proteins transcription initiation complex RNA pol II 5’ 3’ Enhancer-2 Enhencer-1 gene Enhancer-3 -10kb to -50kb -200 TATA box +10kb to +50kb

in structure to differentiate definitely in space and function. promoter enhancer gene  The enhancer always interlinks or overlaps with the promoter.  Sometimes the enhancer and the promoter too close in structure to differentiate definitely in space and function.  The DNA structure of both the promoter and the enhancer is called space-time special promoter. space-time special promoter

Control of gene expression  The action of enhancer is not strict specific. enhancer regulatory protein promoter transcription initiation complex 1 2 n

  direction.  The action of enhancer does not have strict promoter gene 5’ 3’ GCGA…GCT ACGT...ACG enhancer TCG...AGCG GCA...TGCA  

4.4.1.3 silencer The silencers are the negative elements in gene transcription regulation, in contrast to the enhancers. The gene transcription is suppressed when specific regulation protein have bound with the silencers. Some silencers play the role of enhancers sometime this mainly depends to the character of regulation proteins in the nucleolus.

4.4.2 transcription regulation factors The transcription regulation factors are also called transcription factors (TF). Most transcription regulation factors are trans-acting factors, a few ones are cis-acting proteins.

Control of gene expression TATA box various control element -200 silencer +10kb to +50kb enhancer -10kb to -50kb gene 5’ 3’ RNA pol II transcription initiation complex Control of gene expression Trans-acting factor Gene regulatory proteins Gene regulatory proteins

4.4.2.1 type of transcription factors 4.4.2.1.1 general transcription factors It is necessary for the RNA polymerase bind with the promoter. It decides RNA transcription type . It is also regarded as a component of RNA polymerase.

Various transcription factors of RNA polymerase II TF Molecular weitht, kD Function TF II A 12, 19, 35 stabilize TF II D TF II B 33 accelerate that RNA pol II binding with DNA. TF II D TBP/38, TAP/15-250 recognize TATA box TF II E 57(), 34() ATPase TF II F 30, 74 helicase TF II I 120 accelerate that TF II D binding TATA TF II H 35-89 phosphorylation of carboxyl terminal domain of RNA pol II large subunit

4.4.2.1.2 special transcription factors It is necessary for individual gene transcription. It decided the specificity of gene transcription in the time and the space. Most special transcription factors are transcription activators, a few ones are transcription inhibitors.

Most transcription activators are the proteins binding with the enhancer. Most transcription inhibitors are the proteins binding with the silencer. Some transcription inhibitors don’t directly interact with DNA, but bind with the general transcription factor II-D or some transcription activators and decrease effective concentration of the latter in cell and suppress gene transcription.

4.4.2.2 structure of transcription factor Usually, a transcription factor contains a DNA binding domain and a transcription activation domain. Many transcription factors also contain a domain which mediate protein-protein interaction, for example dimerization domain.

4.4.2.2.1 DNA binding domain  It is consist of 60~ 100 amino acid residues usually.  the most familiar structures:  zinc finger structure  basic - helix structure  other structures:  basic leucine zipper structure，bZIP  basic helix-turn helix structure，bHTH  basic helix-loop-helix structure，bHLH

F L + Y zinc finger in TF III A zinc finger in SP1（TF） Zn H Y L F COOH H2N zinc finger in SP1（TF） bind with GC box， discovered the zinc finger early

Zn

basic - helix domain(bAH) DNA binding domain of CTF1 (transcription factor)

basic leucine zipper，bZIP COOH NH2 1 8 15 22 29 36 Leucine residue C N O H CH2 CH CH3

basic helix-turn helix，bHTH 3.4nm 2 1 3 + -phage Cro

basic helix-loop-helix，bHLH 2 1 3

4.4.2.2.2 transcription activation domain  It is usually consist of 30100 amino acid residues .  It is divided three types as follows:  acidic activation domain  glutamine-rich domain  proline-rich domain

4.4.2.2.3 dimerization domain It is the basic leucine zipper or basic helix loop helix in protein structure.

the structure of gene in eukaryote, cis-acting element and trans or cis -acting factor exon intron ATG TAA Poly A site AATAAA, 30bp downstream enhancer terminator silencer initiation site promoter upstream coding strand template strand 5’ 3’ 1 2 n n-1 TATA box CAAT box GC box cis-acting elements trans or cis-acting factors

3.3.3 mRNA transcription activation and regulation gene TATA enhancer 3.3.3 mRNA transcription activation and regulation 5’ 3’ promoter region EBP TF II A pol II TF II F TBP TAF TF II E TF II B

enhancer pol II / TF II F enhancer 5’ pol II TAF TBP 3’ gene TATA TAF TF II A pol II / TF II F PIC TBP TF II B TF II E TIC EBP enhancer gene TATA enhancer 5’ 3’ promoter region EBP pol II TF II F TBP TAF TF II A TF II B TF II E

multilevel regulation of gene expression RNA dsDNA supersolenoid chromatid chromosome solenoid chromatin nucleosomes Transcription bubble activity of gene structure 2 1 3 4 5 6 8 9 10 11 AAAAA hnRNA 7 protein precursor rRNA rRNA Mature tRNA tRNA Mature 12 mature 13 mRNA Mature 14 16 amino acid polysome 15 function multilevel regulation of gene expression

练 习 题 四 1 基因表达的产物可以是 A DNA B tRNA C mRNA D rRNA E 蛋白质 F hnRNA

2 基因表达的组织特异性可表现为 A 不同基因在同一组织中表达不同 B 同一基因在不同组织中表达不同 C 不同基因在不同组织中表达不同 D 不同基因在同一组织中表达相同 E 同一基因在不同组织中表达相同

3 直接参与乳糖操纵子调控的因素是 A I基因编码蛋白 B Z基因编码蛋白 C CAP D Y基因编码蛋白 E 乳糖

4 参与原核基因表达调控的因素是 A 激活蛋白 B 阻遏蛋白 C 某些小分子化合物 D 基本转录因子

5 基因表达调控可以发生在 A 转录起始水平 B 转录水平 C 转录后加工水平 D 翻译起始水平 E 翻译水平 F 翻译后加工水平 G 复制水平

6 顺式作用元件是下述的 A TATA盒和 CCAAT盒 B 具有调节功能的各种DNA序列 C 具有调节功能的 5’ 侧翼序列 D 具有调节功能的 3’ 侧翼序列 E 所有非编码序列

7 反式作用因子是 A 转录调节蛋白 B 转录调节因子 C RNA聚合酶 D DNA聚合酶 E DNA酶 I

8 真核基因表达调控的特点是 A 正性调节占主导 B 伴有染色体结构变化 C 转录与翻译分隔进行 D 转录与翻译偶联进行 E 负性调节占主导

9 乳糖操纵子 A 在没有半乳糖存在时处于阻遏状态 B 在半乳糖存在时可能处于表达状态 C 有高浓度半乳糖、低浓度葡萄糖存在时处于 表达状态 D 有半乳糖存在时处于阻遏状态 E 没有半乳糖存在时处于表达状态

10 可影响RNA聚合酶活性的因素是 A 启动子或启动序列 B 调节蛋白的性质 C RNA转录本的结构 D RNA转录本的长度 E 多聚A序列的长度

11 基因表达具有 A 阶段特异性 B 组织特异性 C 细胞特异性 D 时间特异性 E 空间特异性

12 原核基因表达调控的意义是（只有一个正确答案） A 调节生长与分化 B 调节发育与分化 C 调节生长、发育与分化 D 调节代谢，适应环境 E 维持细胞特性和调节生长

13 真核基因表达调控的意义是（只有一个正确答案） A 调节代谢，维持生长 B 调节代谢，维持发育与分化 C 调节代谢，发育与分化 D 调节代谢，适应环境 E 调节代谢，适应环境，维持生长、发育与分化

14 下述关于管家基因表达的描述最确切的是 （只有一个正确答案） A 在生物个体的所有细胞中表达 B 在生物个体生命全过程几乎所有细胞中持续表达 C 在生物个体生命全过程部分细胞中持续表达 D 特定环境下， 在生物个体生命全过程几乎所有细 胞中持续表达 E 特定环境下，在生物个体生命全过程部分细胞中 持续表达

15 基本的基因表达（只有一个正确答案） A 有诱导剂存在时表达水平增高 B 有诱导剂存在时表达水平降低 C 有阻遏剂存在时表达水平增高 D 有阻遏剂存在时表达水平降低 E 极少受诱导剂或阻遏剂的影响

16 紫外线照射引起DNA损伤时，细菌DNA修复酶 基因表达增强，这种现象被称为（只有一个正确答案） A 诱导 B 阻遏 C 基本的基因表达 D 正反馈 E 负反馈

17 大多数基因表达调控的最基本环节是（只有一个 17 大多数基因表达调控的最基本环节是（只有一个 正确答案） A 复制水平 B 转录水平 C 转录起始水平 D 转录后加工水平 E 翻译水平

18 顺式作用元件的最确切定义是（只有一个正确答案） A TATA盒和 CCAAT盒 B 具有调节功能的各种DNA序列 C 具有调节功能的 5’ 侧翼序列 D 具有调节功能的 3’ 侧翼序列 E 所有非编码序列

19 对乳糖操纵子来说（只有一个正确答案） A CAP是正性调节因素，阻遏蛋白是负性调节因素 B CAP是负性调节因素，阻遏蛋白是正性调节因素 C CAP和阻遏蛋白都是正性调节因素 D CAP和阻遏蛋白都是负性调节因素 E 在不同条件下，CAP和阻遏蛋白均显示正性 或负性调节特点

20 一个操纵子通常含有（只有一个正确答案） A 一个启动序列和一个编码基因 B 一个启动序列和数个编码基因 C 数个启动序列和一个编码基因 D 数个启动序列和数个编码基因 E 一个启动序列和数个调节基因

第十四章 基因重组与基因工程 第二节 DNA重组技术

一、 DNA重组技术相关概念 （一） DNA克隆 （二） 工具酶 （三） 目的基因 （四） 基因载体

（一） DNA克隆 所谓的DNA克隆就是DNA分子扩增的过程。 通常首先将某一目的DNA片段插入一适当的载 体中，构建重组复制子，继而转化宿主细胞并 筛选出含有重组复制子的细胞，然后进行扩增 即可以获得大量的目的DNA分子。上述过程即 为DNA克隆。

（二）DNA重组技术相关工具酶 工 具 酶 功 能 限制性核酸内切酶：识别特异DNA序列并切割之 工 具 酶 功 能 限制性核酸内切酶：识别特异DNA序列并切割之 DNA连接酶： 缝合DNA分子上的单链切口，使两个线型DNA 分子的匹配粘端相互连接在一起。 DNA聚合酶 I：  填补3’凹端，  DNA测序，  合成cDNA 的第二条链，  缺口平移制作高比活探针 。 反转录酶：  合成cDNA的第一条链，  替代DNA聚合酶 I 填补3’凹端，制作单链DNA探针，DNA测序 多聚核苷酸激酶： 催化多聚核苷酸5’羟基末端磷酸化以标记探针。 末端转移酶： 在DNA 的3’羟基端进行同质多聚物加尾。 碱性磷酸酶： 去除线型DNA 分子5’末端的磷酸基。 （二）DNA重组技术相关工具酶

限制性核酸内切酶特异性酶切位点的结构特点 限制性内切酶 辨认的序列及其切割位点 BamH I EcoR I GAATTC CTTAAG GGATCC CCTAGG 5’ Pvu I Sst I GAGCTC CTCGAG CGATCG GCTAGC Alu I Sma I AGCT TCGA CCCGGG GGGCCC

（三） 目的基因（target gene） 所感兴趣的基因就是目的基因。

目的基因的来源 直接来源于基因组，适合于原核基因。 来源于人工合成，适合于小基因。 来源于mRNA，适用性强。 来源于cDNA文库或基因组文库。

（四） 基因载体 所谓的基因载体是一些经过结构改造的DNA 分子。他们可以携带目的基因或DNA片段，并实 现后者的克隆扩增甚至是表达。

质粒 pBR322 tetr 4.36kb 三个基本条件 ori ampr 一个复制起始点，a origin Xmn I 3966 2034 Xmn I Pst I 3612 2067 Pvu II 1424 Ava I 650 Sal I 375 BamH I 质粒 pBR322 4.36kb 29 Hind III EcoR I 0 三个基本条件 一个复制起始点，a origin 一个筛选基因，a screening gene 单一限制性酶切位点，a single restriction site tetr ampr ori 氨苄青霉素抗性基因 四环素抗性基因

分 选、切 连 转 筛、表达 二、 DNA重组技术基本原理 （一） 目的基因的分离 —— （二） 克隆载体的选择—— （三） 目的基因与载体的连接—— （四） 重组子的转化—— （五） 重组子的筛选 （六） 目的基因的表达 筛、表达 分 选、切 连 转

分、选、切、连 genome DNA plasmid Hpa II Ligase recombinant CGG C GGC CCGG

vectors and recombinants 重组子的转化 vectors and recombinants competent cells

重组子的筛选 氨苄青霉素 + 质粒提取 限制性内切酶特异切割 1 2 DNA标准分子 -

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