第十八讲- 北大生命科学学院.ppt

1、,第十章 基因和发育,By Hongwei Guo, Peking University, 2008.12.24,第三讲 动物发育的基因调控 Fruit fly embryo development,Development,In multicellular organisms, life begins as a single cell (zygote). With few exceptions, somatic cells contain the same genetic information as the zygote. In development, cells commit to sp

2、ecific fates and differentially express subsets of genes. Cells identify and respond to their positions in developmental fields. Most developmental decisions involve changes in transcription.,Pattern formation （形态建成） Is the development of a spatial organization of tissues and organs Pattern formatio

3、n in animals and plants results from similar genetic and cellular mechanisms Occurs continually in plants，but is mostly limited to embryos and juveniles in animals,Positional information,Consists of molecular cues that control pattern formation Tells a cell its location relative to the bodys axes an

4、d to other cells,Location, location, location!,Temporal information: e.g. CO, FT, FLC,Timing, timing, timing!,Positional information,Localization of mRNAs/proteins within zygote establishes positional information -where developmental fields begin as a single cell Formation of concentration gradients

5、 of extracellular diffusible molecules establishes positional information in multicellular developmental fields works by signal transduction diffusible molecules are known as morphogens,Cytoplasmic determinants,Differential induction,cytoskeletal elements,Polarity determination（极性确定）,Pattern formati

6、on was extensively studied in the model organism Drosophila melanogaster-Fruit fly,The fruit fly is small and easily grown in the laboratory. It has a generation time of only two weeks and produces many offspring (up to 300). Embryos develop outside the mothers body. Sequencing of Drosophila genome

7、was completed in 2000.,Drosophila as a genetic model,Isolation and characterization of developmental mutants,Nusslein-Volhard and Wieschaus concentrated their efforts on understanding early embryogenesis, they looked for recessive embryonic lethal mutations, and classified them according to their ph

8、enotype before death. Lewis did pioneering research on late embryogenesis, who discovered homeotic mutants - mutant flies in which structures characteristic of one part of the embryo are found at some other location.,果蝇发育的时间周期取决于温度，在25下约9-14天为一个周期，其中第一天为胚胎发育期。幼虫经历三个阶段，到第四天蜕皮分化后蛹化，在蛹中经过5天的变态，再发育为成虫。另

9、外，还应加上受精所需的1-2天，共约两周左右。成年果蝇存活约9天。,Key developmental events in life cycle of Drosophila,Follicle cell,Nucleus,Egg cell,Fertilization,Nurse cell,Egg cell developing within ovarian follicle,Laying of egg,Egg shell,Nucleus,Fertilized egg,Embryo,Multinucleate single cell,Early blastoderm,Plasma membrane

10、formation,Late blastoderm,Cells of embryo,Yolk,Segmented embryo,Body segments,0.1 mm,Hatching,Larval stages (3),Pupa,Metamorphosis,Head,Thorax,Abdomen,0.5 mm,Adult fly,Dorsal,Anterior,Posterior,Ventral,BODY AXES,卵母细胞(oocyte)自身的细胞核不具转录活性，而由母源抚育细胞(nurse cells)、滤泡细胞(follicle cells) 利用自身的基因和细胞资源提供遗传信息和营

11、养物质，然后输入到卵母细胞中。这些被输入到卵母细胞的基因被称为母源影响基因（maternal effect genes），对卵子的发育有重大影响。,Bicoid mRNA, Nanos mRNA是nurse cell分泌进入卵母细胞，前者与RNA binding protein swallow, exuperantia 结合后在microtubule作用下被铆钉在卵细胞前端； Nanos mRNA则是与RNA binding protein tudor, oskar等结合被运动到了后端。此过程发生在卵细胞受精之前。,Early syncitial(合胞体)development zygotic

12、 nucleus divides 9 times with no cell division some nuclei migrate to posterior pole to give rise to germ line 4 more mitotic divisions without cell division,Drosophila embryo development (1),Drosophila development (2),At 10 hours, 14 segments 3 head 3 thoracic 8 abdominal,At 12 hours, organogenesis

13、 begins At 24 hours, larva hatches,Maternal effect genes Encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila,Axis Establishment,Maternal effect genes,bicoid, nanos, hunchback, caudal,Drosophila larvae with wild-type and bicoid mutant phenotypes. A mutation

14、 in the mothers bicoid gene leads to tail structures at both ends (bottom larva). The numbers refer to the thoracic and abdominal segments that are present.,Phenotype of bicoid,Embryos derived from bicoid(bcd) females lack head and thorax. Cytoplasmic transplantation experiment reveal that BCD activ

15、ity is localized at anterior pole of wide-type embryos.BCD activity can induce anterior development in mutant embryo at any position along antero-posterior axis and suppress posterior development.,WT,bcd,bcd injected with cytoplasm,Hans Georg Frohnhfer Bicoid transcripts were concentrated in the cor

16、tical cytoplasm in the form of an anterior cap; accumulate at the anterior margin of oocytes in the female abdomen; after fertilization transcripts move posteriorly, forming a concentration gradient along the anteroposterior axis in cleavage-stage embryos; and during nuclear cycle 14, transcripts di

17、splay an extended gradient along the apical cortex of the syncytial-blastoderm embryo. Bicoid mRNA is transported to the apical nuclear periplasm during syncytial blastoderm; and bcd transcripts are degraded rapidly during the first third of nuclear cycle 14; Bcd protein gradient is dictated by the

18、bcd mRNA gradient and its subsequent translation; Bicoid mRNA colocalizes with Staufen (Stau), suggeting that the Bicoid mRNA gradient forms by transport of a Stau-bcd mRNA complex.,Gradients of bicoid mRNA and Bicoid protein in normal egg and early embryo,Formation of the bicoid morphogen gradient:

19、 an mRNA gradient dictates the protein gradient,(C) High concentrations of Bicoid activate the transcription of orthodenticle in the anterior of the embryo. (D) hunchback transcription is activated at a lower threshold concentration of Bicoid, and it is therefore expressed throughout the anterior ha

20、lf of the embryo. (Note that the posterior stripe of hunchback is under separate regulation from the terminal system.) (E) Bicoid represses the translation of caudal mRNA to produce a posterior to anterior gradient of Caudal protein.,How the Bicoid Gradient Works?,RNA binding and translational suppr

21、ession.,mRNA,protein,protein in bcd,Bicoid dependent formation of caudal protein gradient,Bicoid protein (89154aa) can bind to Caudal mRNA (19192018),Bicoid protein suppresses translation of Caudal 3UTR containing mRNA.,Caudal 3UTR,Bicoid associates with the 5-cap-bound complex of caudal mRNA and re

22、presses translation,Autoradiogram showing that in vitro translated 35S-labeled BCD (input; a schematic representation of the protein is shown at the bottom of e) is capable of interacting with m7GTP-sepharose-bound recombinant eIF4E in the presence of in vitro transcribed cad 3-UTR,四种母源影响基因的mRNA和蛋白沿

23、果蝇卵子和胚胎前-后轴分布的浓度变化图,A,P,BICOID,CAUDAL,HUNCHBACK,NANOS,Drosophila anterior-posterior axis,Determined by gradients of BCD (product of bicoid) and HB-M (product of hunchback) BCD mRNA maternally deposited in anterior BCD mRNA tethered to “” ends of microtubules via 3 UTR HB-M protein gradient depends o

24、n NOS protein nos mRNA tethered to “+” end of microtubule via 3 UTR NOS protein gradient blocks translation of hb-m mRNA, resulting in HB-M gradient opposite gradients of BCD and Nos determine A-P axis (Nos is a RNA binding protein that represses translation of HB),Developmental fate determined thro

25、ugh transcription factor interactions Sequential activation of three sets of segmentation genes,1.Gap genes,2.Pair-rule genes,3.Segment polarity genes,Drosophila development (3)- Segmentation Pattern -,Segmentation genes Produce proteins that direct formation of segments after the embryos major body

26、 axes are formed,(segment identity genes),Gap genes map out the basic subdivisions along the A-P axis.,Mutations cause “gaps” in segmentation Kruppel (Kr), knirps (Kni) and hb-Z promoters have differential sensitivity to BCD and/or HB-M Bifurcation of development: targets of gap gene encoded transcr

27、iption factors one branch to establish correct number of segments one branch to assign proper identity to each segment,Gap gene expression -position-dependent sets of (I)transcriptional activator/repressor and (II)post-translational protein turn over systems,Occurs in discrete domains and defines sp

28、ecific, overlapping sets of segment primordia. Their protein products form broad and overlapping concentration gradients which are controlled by maternal factors and by mutual interactions between the gap genes themselves. Once established, these overlapping gap protein gradients provide spatial cue

29、s which generate the repeated pattern of the subordinate pair-rule gene expression.,Pair-rule genes define the modular pattern in terms of pairs of segments. Mutations result in embryos with half the normal segment number.,Segment polarity genes set the anterior-posterior axis of each segment. Mutat

30、ions produce embryos with the normal segment number, but with part of each segment replaced by a mirror-image repetition of some other part.,Region-specific combinations of different gap genes eventually generate the periodic pattern of pair-rule gene expression by the direct interaction with indivi

31、dual cis-acting stripe elements of particular pair-rule gene promoters. primary pair-rule genes (even-skipped, hairy, and runt) respond directly to gap information, they regulate the expression of the remaining so-called secondary pair-rule genes (fushi-tarazu, odd-skipped, paired, odd-paired, and s

32、loppy-paired), which in turn play a more direct role in the establishment of segment-polarity gene expression. Pair-rule patterning, producing seven stripes, is a result of the action of gap genes, whose function is to define each of the seven stripes of primary pair-rule genes. pair-rule genes regu

33、late engrailed (en) and wingless-the segment-polarity gene, resulting in the creation of the necessary fourteen evenly spaced segments on either side of the segmental border. A well-characterized target of pair-rule function is engrailed (en), which is expressed in fourteen stripes. Correct establis

34、hment of the fourteen engrailed stripes requires the activities of all the pair-rule genes. However, in general, mutations in individual pair-rule genes affect either odd- or even-numbered engrailed stripes.,The pair-rule genes are expressed in striped patterns of seven bands(even-skipped) perpendic

35、ular to the anterior-posterior axis. They regulate the engrailed genes transcription. Cells that make Engrailed can make the cell-to-cell signaling protein Hedgehog (green in lower picture). Hedgehog is not free to move very far and activates a thin stripe of cells adjacent to the Engrailed-expressi

36、ng cells. Only cells to one side of the Engrailed-expressing cells are competent to respond to Hedgehog because they express the receptor protein Patched (blue in lower picture). Cells with activated Patch receptor make the Wingless protein (red in lower picture). Wingless protein acts on the adjace

37、nt rows of cells by activating its cell surface receptor, Frizzled. Wingless also acts on Engrailed-expressing cells to stabilize Engrailed expression after the cellular blastoderm forms. The reciprocal signaling by Hedgehog and Wingless stabilizes the boundary between each segment. The Wingless pro

38、tein is called wingless because of the phenotype of some wingless mutants. Wingless also functioned during metamorphosis to coordinate wing formation.,Overview,Drosophila Segmentation,Segment number gap gene products activate pair-rule genes several different pair-rule genes expression produces repe

39、ating pattern of seven stripes, each offset pair-rule products act combinatorially to regulate transcription of segment-polarity genes expressed in offset pattern of 14 stripes Segment identity gap gene products target cluster of homeotic gene complexes encode homeodomain transcription factors mutat

40、ions alter developmental fate of segment e.g., Bithorax (posterior thorax and abdomen) and Antennapedia (head and anterior thorax),Segment Identity,The identity of Drosophila segments is set by master regulatory genes called homeotic genes,homeotic transformation -Structures characteristic of a part

41、icular part of the animal arise in the wrong place.,bithorax,Drosophila homeotic loci，whose genes determine the identity of body structures.,All homeotic genes of Drosophila include a 180-nucleotide sequence called the homeobox, which specifies a 60-amino-acid homeodomain, part of a transcription fa

42、ctor.,interesting correlation,The homeodomain is responsible solely for binding to DNA. Proteins containing homeodomains may be either activators or repressors of transcription.,The activator or repressor domains both act by influencing the basal apparatus. Activator domains may interact with coacti

43、vators that in turn bind to components of the basal apparatus. Repressor domains also interact with the transcription apparatus. The repressor Eve, for example, interacts directly with TFIID.,Gene products in Dm embryo development,Many are homeodomain-containing transcription factors,Homeobox-contai

44、ning genes often called Hox genes in mammals conserved in animals for hundreds of millions of years. Related sequences are present in yeast, animals (including human), but not in plants. MADS box genes in plants (in flower pattern formation),An identical or very similar nucleotide sequence in the ho

45、meotic genes of both vertebrates and invertebrates,Not all homeobox-containing genes are homeotic genes that are associated with development. some dont directly control the identity of body parts.,Drosophila dorsal-ventral axis,Determined by gradient of transcription factor DL (encoded by dorsal) gr

46、adient established by interaction of spz and Toll gene products deposited in oogenesis and released during embryogenesis SPZ-TOLL complex triggers signal transduction pathway in cells that phosphorylates inactive DL Phosphorylated DL migrates to nucleus, activating genes for ventral fates,sptzle is

47、a maternal effect gene,The sptzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo.,Generalizations,Asymmetry of maternal gene products establishes positional information used for early development Successive rounds of e

48、xpression of genes encoding transcription factors establish axes and body part identity Positive feedback loops maintain/stabilize differentiated state Differences in types and concentrations of transcription factors result in different outputs,Developmental parallels,Early animal development follow

49、s fundamentally similar pattern Remarkable similarity among homeotic genes one HOM-C cluster in insects four HOX clusters in mammals paralogous to insect cluster expressed in segmental fashion in early development,Comparison of Animal and Plant Development,In both plants and animals Development reli

50、es on a cascade of transcriptional regulators turning genes on or off in a finely tuned series But the genes that direct analogous developmental processes Differ considerably in sequence in plants and animals In plants, Homeobox-containing genes do not function in pattern formation as they do in ani

51、mals MADS-box containing genes do this part,第十一章 基因组学与比较基因组学,By Hongwei Guo, Peking University, 2009.12.28,Genomics and Comparative Genomics,当代生物学已进入基因组 (Genome)时代,现代生物学的主要研究对象就是基因What？ How？ Why？,基因组计划,基因组（genome）是生物体内遗传信息的集合，是某个特定物种细胞内全部DNA分子的总和。 基因组学（genomics）是指研究并解析生物体整个基因组的所有遗传信息的学科。 基因组计划（Genom

52、e Project）是指对人类以及其它生物体全基因组的测序工作(sequencing)。 人类基因组计划（Human Genome Project，HGP）： 90年代提出并已基本完成，同40年代原子弹爆炸，60年代人类登月一起被认为是二十世纪科技发展史上的三大创举。,人类基因组计划,基因组计划（Genome Project）是指对人类以及其它生物体全基因组的测序工作(sequencing)。 人类基因组计划（Human Genome Project，HGP）： 90年代提出并于2000年基本完成，同40年代原子弹爆炸，60年代人类登月一起被认为是二十世纪科技发展史上的三大创举。,Celera

53、: Craig Venter,Intl. Cons: Francis Collins,人类基因组计划的目的,解码生命、了解生命的起源、了解生命体生长发育的规律、认识种属之间和个体之间存在差异的起因、认识疾病产生的机制以及长寿与衰老等生命现象、为疾病的诊治提供科学依据。,回答了What的问题,6国科学家组成的国家人类基因组中心主要研究比例,美国：WASHMIT等7家研究中心，贡献率为54。 英国：SANGER一家研究中心，贡献率为33。 日本：RIKEN等两家研究中心，贡献率为7。 法国：GENOSCOPE研究中心，贡献率为2.8。 德国：IMB等3家研究中心，贡献率为2.2。 中国：北京华大研

54、究中心、国家南北方基因研究 中心等三家，贡献率为1。,2000年6月 白宫的庆典,Venter,Collins,Public effort- strategy:,Celera - strategy:,Sequencing Strategies,Celeras view of International Consortium,International Consortiums view of Celera,Unfair competition: IC delivering the same goods but with state funding.,Unfair competition: Cel

55、era delivering the same goods but can use IC data, while IC cannot use Celera data.,遗传图谱(Genetic Map)vs 物理图谱 (Physical Map),遗传图又称为连锁图（Linkage Map），是指基因或DNA标志在染色体上的相对位置（或距离），通常以基因或DNA片段在染色体交换过程中的分离频率（cM）来表示。cM值越大，两者之间遗传距离越远。 物理图谱是指以已知序列的DNA片段（DNA marker)在染色体上的实际位置，位点之间的距离（图距）以碱基对（bp，kb，Mb）作为测量单位的基因组图

56、。,DNA遗传标记 (DNA marker),第一代DNA遗传标记是RFLP（Restriction Fragment Length Polymorphism）。DNA序列上的微小变化，甚至1个核苷酸的变化，也能引起限制性内切酶切点的丢失或产生，导致酶切片段长度的变化。 第二代DNA遗传标记SSLP（Simple Seqeuce Length Polymorphism)利用了存在于人类基因组中的大量重复序列，包括重复单位长度在15-65个核苷酸左右的小卫星DNA（minisatellite DNA），重复单位长度在2-6个核苷酸之间的微卫星DNA（microsatellite DNA）。 第三

57、代DNA遗传标记SNP （ single nucleotide polymorphism） ，也是最广泛的遗传标记，是分散于基因组中的单个碱基的差异。这种差异包括单个碱基的缺失和插入，但更常见的是单个核苷酸的替换，即单核苷酸的多态性。 到目前为止已经在人类基因组发现了超过1000万个SNP位点，平均每300bp中就有一个SNP！,RFLP marker,SSLP markers,WT,mut,SNP marker,Single nucleotide polymorphisms (SNPs) are highly abundant, and are estimated to occur at 1

58、 out of every 1,000 bases in the human genome. SNPs in the coding regions of genes that alter the function or structure of the encoded proteins are a necessary and sufficient cause of most of the known recessively or dominantly inherited monogenic disorders. These SNPs are routinely analysed for dia

59、gnostic purposes. However, most SNPs are located in non-coding regions of the genome, and have no direct known impact on the phenotype of an individual. These SNPs are useful as markers in population genetics and evolutionary studies.,SNP detection or genotyping,The panels illustrate detection of the A-allele of an A-to-G transition. The G-allele would be detected analogously in a parallel reaction. In panel a, hybridization with allele-specific oligonucleotides (ASOs) is shown. Two short ASO probes are used, usually with the nucleotide complementa