《線粒體基因組》PPT課件.ppt

1、Mitochondrial molecular genetics,Mitochondrial molecular genetics,Mitochondria are the main site of ATP synthesis in eukaryote cells and as such are vital for the health and survival of the cell They are also one of the sites at which apoptosis is mediated,These lectures will explore the molecular g

2、enetics of mitochondria, how they are made, the structure of their genome, how they evolved , and how mitochondrial gene expression is controlled.,Mitochondrial molecular genetics focus on mitochondria: brief overview of their function and structure mtDNA structure and replication: - animals - yeast

3、 - plants inheritance of mitochondria - petite mutants of yeast biogenesis of mitochondria by fission,MITOCHONDRIA essential for cell life - ATP synthesis - many metabolic intermediates, essential for cell death - unprogrammed death: necrosis ( eg, due to loss of energy status) - programmed cell dea

4、th (apoptosis - controlled cell destruction), Two membranes Inner membrane invaginated Numbers of mitochondria per cell vary but usually 100s/cell,Matrix contains the TCA cycle (and other) soluble enzymes Inner membrane contains metabolite transporters and the electron transport chain,Mitochondrial

5、structure,The ribosomes can actually be visualized in some mitochondria. In these figures, they are seen in the matrix as small dark bodies. DNA can also be visualized in mitochondria. The DNA is circular and resembles that of a bacterium in its basic structure.,Mitochondria also have their own ribo

6、somes and tRNA: 22 tRNAs rRNAs (16S and 12S),Mitochondria contain DNA molecules with an assortment of genes. Mitochondrial genetic system consist of DNA and the molecular machinery needed to replicate and express the genes contained in this DNA. This machinery includes the macromolecules needed for

7、transcription and translation. Mitochondria even possess their own ribosomes. Many of these macromolecules are encoded by mitochondrial genes, but some are encoded by nuclear genes and are therefore imported from the cytosol.,Mitochondria have their own DNA and Ribosomes Mitochondria have some of th

8、eir own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix in structures called nucleoids. Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA).,mitochondrial DNA,Mitochondrial DNA (mtDNA),mtDNA was discovered in the 1960s; revealed a

9、s DNA-like fibers within mitochondria. The complete nucleotide sequences of mtDNA molecules from many different species have now been determined. mtDNA vary enormously in size, from about 6kb in Plasmodium to 2500 kb in some of the flowering plants. Each mitochondrion appears to contain several copi

10、es of DNA.,Mitochondrial DNA (mtDNA),In a vertebrate oocyte, for example, it has been estimated that as many as 100 million copies of the mtDNA are present. Somatic cells, however, have fewer copies, perhaps less than 1 000. Most mtDNA molecules are circular, but in some species, such as alga Chlamy

11、domonas reinhardtii 萊茵衣藻 , they are linear. In the vertebrates 37 distinct genes are packed into a 16 to 17-kb circle leaving little or no space between genes.,MITOCHONDRIAL DNA (YELLOW) IN THE UNICELLULAR ORGANISM EUGLENA GRACILIS. THE NUCLEAR DNA (RED) IS ALSO VISIBLE.,Plant mtDNA,In some of the f

12、lowering plants an unknown number of genes are dispersed over a very large circular DNA molecule hundreds or thousands of kilobase in size. In these plants the mitochondrial genes may become separated onto different circular molecules by a process of intramolecular recombination.,Plant mtDNA,This re

13、combination is mediated by repetitive sequences located in the mtDNA. An exchange between two of the repetitive sequences can partition the “master” mtDNA circle into two smaller circle, a process that superficially resembles the excision of a lambda prophage from E. coli chromosome. In some species

14、, several DNA circles of different sizes are formed by recombination between pairs of repetitive sequences located at different positions around the master DNA circle. These molecules is difficult to study, and more research is needed to elucidate the mechanism that produces them.,Intramolecular rec

15、ombination in the mtDNA of the Chinese cabbage, Brassica campestris油菜. Recombination between the repeated elements in the large circular DNA molecule partitions this molecule into two smaller ones. Alternatively, the repeated elements in the two small molecules may recombine with each other to produ

16、ce a single large molecule.,The structure of mtDNA,The structure of mtDNA molecules has been studied by DNA sequencing. Animal mtDNA is small and compact. In human beings, for example, the mtDNA is 16,659 base pairs long and contains 37 genes, including two that encode ribosomal RNAs, 22 encode tran

17、sfer RNAs, and 13 that encode polypeptides involved in oxidative phosphorylation, the process that mitochondria use to recruit energy. In mice, cattle, and frogs, the mtDNA is similar to that of human beings an indication of a basic conservation of structure within the vertebrate subphylum.,Map of h

18、uman mtDNA showing the pattern of transcription. Genes on the inner circle are transcribed from the L strand of the DNA, whereas genes on the outer circle are transcribed from the H strand of the DNA. Arrows show the direction of transcription. ND1-6 are genes encoding subunits of the enzyme NADH re

19、ductase; the tRNA genes in the mtDNA are indicated by abbreviations for the amino acids.,The structure of mtDNA,Invertebrate mtDNA is about the same size as vertebrate mtDNA, but it has a somewhat different genetic organization. These differences seem to have been caused by structural rearrangements

20、 of the genes within circular mtDNA molecule.,The structure of mtDNA,In fungi, the mtDNA is considerably larger than it is in animals. Yeast, for example, possesses circular mtDNA molecules 78 kb long. These molecules contain at least 33 genes, including 2 that encode ribosomal RNAs, 23 to 25 that e

21、ncode transfer RNAs, 1 that encodes a ribosomal protein, and 7 encode different polypeptides involved in oxidative phosphorylation. The yeast mtDNA is larger than animal mtDNA because several of its genes contain introns and there are long noncoding sequences between some of the genes. Animal mtDNA

22、does not contain introns.,The structure of mtDNA,Plant mtDNA is much larger than the mtDNA of others organisms. It is also more variable in structure. These conclusions come from crude physical and chemical analysis and from DNA sequencing. One of the first plant mtDNAs to be sequenced is from the l

23、iverwort地錢 , Marchantia polymorpha. The mtDNA from this primitive, nonvascular (非維管) plant is a 186-kb circular molecule with 94 substantial open reading frames (ORFs) , some corresponding to known genes and others having still unassigned genetic functions. The latter ORFs are therefore called URFs,

24、 for unassigned reading frames. 32 distinct introns have been found in the Marchantia mtDNA, accounting for about 20% of the molecule. In vascular (維管) plants, the mtDNA is larger than it is in Marchantia; for example, it is a 570-kb circular molecule in maize and a 300-kb circle in the watermelon西瓜

25、 .,The structure of mtDNA,Higher plant mtDNA molecules contain many noncoding sequences , including some that are duplicated. The actual number of genes per mtDNA molecule is unknown. Physical mapping of some of these genes has shown that they are located in different position in the mtDNA circles o

26、f different species, even when the species are fairly closely related. This implies that mtDNA of higher plants has undergone many genetic rearrangements during its evolution.,Expression of Mitochondrial genes,The simple mtDNA of vertebrates are organized into two large transcription unit, each enco

27、ding the information of several genes. When the two strand of human mtDNA are separated by centrifugation, one proves to be denser the H strand (for heavy), than the other referred as the L (for light). The promoters for the H and L transcription units are situated just upstream of the phenylalanine

28、 tRNA gene. The transcripts are extended in opposite directions around the mtDNA molecule.,The transcript from the H strand encodes 2 ribosomal RNAs, 14 tRNAs, and 12 polypeptides. The transcript from the L strand encodes 8 tRNAs and 1 polypeptide. Each transcript is cleaved to separate the tRNAs fr

29、om the rRNAs and mRNAs, and mRNAs are polyadenylated. Each mRNA is then translated into polypeptides, using the mitochondrial ribosomes and a combination of nuclear and ribosomal tRNAs.,Expression of Mitochondrial genes,Translation in the mitochondria proceeds much as it does on the ribosomes of the

30、 cytosol, except that some of the codons have a different meaning. AGA and AGG are termination codons in mammalian mitochondria, whereas in the cytosol they specify the incorporation of arginine; UGA, which is termination codon in the cytosol, is a tryptophan codon in the mitochondria; and AUA, whic

31、h encodes isoleucine in cytosol, is the methionine initiation codon in the mitochondria.,Expression of Mitochondrial genes,In fungi and plants the mtDNA is organized into many separate transcription units, some containing the information for more than one gene. Little is known about the details of t

32、ranscription, but in yeast, the mitochondrial RNA polymerase is a single polypeptide encoded by a nuclear gene. RNA processing separates plant mitochondrial transcripts into their constituent parts and also removes the introns, which are present in several plant mitochondrial genes. THE MECHANICS OF

33、 THESE EVENTS ARE POORLY UNDERSTOOD.,Expression of Mitochondrial genes,Another peculiarity of plant mitochondrial gene expression is that many of the mtRNA transcripts undergo editing; that is, some of the nucleotides are changed after transcript has been synthesized. The most frequent change is C t

34、o U (occasionally U to C). Thus, RNA editing alters the composition of codons in plant mitochondrial transcript. Editing alters the information that is actually encoded in the mtDNA and allows functional polypeptides to be synthesized. Editing is not found in the nonvascular plants (mosses and algae

35、藻類和苔蘚植物). The editing mechanism probably evolved sometime after plants had become established on the land.,Expression of Mitochondrial genes,Yet a third peculiarity of plant mitochondrial gene expression is that some mitochondrial mRNAs are formed by the process of trans-splicing. It occurs when seg

36、ments of a gene are scattered over the mtDNA molecule. Each gene segment is transcribed independently, and exons of the different transcripts are spliced together by interactions between the introns that flank them.,Expression of Mitochondrial genes,分子內(nèi)(intramolecular) 剪接(cis splicing) 以及分子間(intermo

37、lecular) 剪接(trans splicing),Trans-splicing in wheat mitochondria. Four different RNAs contribute to the final mRNA encoding a polypeptide of the enzyme NDH reductase.,INTERPLAY BETWEEN MITOCHONDRIAL AND NUCLEAR GENE PRODUCTS,Most perhaps all-mitochondrial gene products function solely within mitocho

38、ndrion. However, they do not function alone. Many nuclear gene products are imported to augment or facilitate their function. Many of the polypeptides needed for aerobic metabolism are also synthesized in cytosol (ATPase that is responsible for binding the energy of aerobic metabolism into ATP). How

39、ever, because some of the subunits of this protein are synthesized in the mitochondria, the complete protein is actually a mixture of nuclear and mitochondrial gene products.,INTERPLAY BETWEEN MITOCHONDRIAL AND NUCLEAR GENE PRODUCTS,This dual (雙重的) composition suggests that nuclear and the mitochond

40、rial genetic systems are coordinated in some way so that equivalent amounts of their products are made; possible molecular mechanisms for this coordination are currently under investigation.,KEY POINTS,mtDNA molecules range from 6-kb to 2500-kb in size, and most of them appear to be circular. mtDNA

41、molecules contain genes for some of the ribosomal RNAs, transfer RNAs, and polypeptides used within the mitochondrion. The structure, organization, and expression of mitochondrial genes vary among species. In some organisms, the transcripts of mitochondrial genes are edited after they are synthesize

42、d. Both mitochondrial and nuclear gene products are needed for normal mitochondrial function.,mt DNA and human disease,Recent research has demonstrated that several human diseases are caused by mitochondrial defects, and in some cases, these defects are due to mutations in the mtDNA. One such diseas

43、e is Lebers hereditary optic neuropathy (LHON), a condition characterized by the sudden onset of blindness in adults. This disease is associated with the death of the optic nerve (at a physiological level), and with mutation in any of several mitochondrial genes (at a molecular level). Each mutation

44、 changes an amino acid in one of the mitochondrial proteins reducing the efficiency of oxidative phosphorylation. The reduction is great enough to destroy the function of the optic nerve and cause total blindness. It is not known why this effect is limited to the optic nerve. LHON is inherited stric

45、tly through the maternal line.,Another disorder caused by a mutation in the mtDNA is a Pearson marrow-pancreas syndrome, characterized by a loss of bone-marrow cells during childhood, is frequently fatal. It is caused by a fairly large deletions in the mtDNA. People with this syndrome almost never h

46、ave affected parents. Thus, the causative deletion probably occurs spontaneously during development in the child or during oogenesis in the mother. Individuals with Pearson syndrome actually have a mixture of deleted and normal mtDNA an example of mitochondrial heteroplasmy. Homoplasmic individuals

47、have never been observed.,mt DNA and human disease,The molecular genetics ofChloroplasts,Chloroplasts contain DNA molecules with an assortment of genes. Chloroplast are specialized forms of a general class of plant organelles called plastids質(zhì)體 . Botanists distinguish among several kinds of plastids,

48、 including chloroplasts (plastids containing pigments), amyloplasts (plastids containing starch造粉體：一種形成淀粉的植物性白色體), and elaioplasts (油質(zhì)體 plastids containing oil or lipid). All three types seem to develop from small membrane-bounded organelles called proplastids前質(zhì)體, and, within a particular plant spec

49、ies, all seem to contain the same DNA. This DNA is generally referred to as chloroplast DNA, abbreviated simply as cpDNA.,CHLOROPLAST DNA,In higher plants, cpDNA typically range from 120 to 160 kb in size, and in algae, from 85 to 292 kb. In a few species of green algae the cpDNA is much larger, abo

50、ut 2000 kb. The cpDNA seems to be organized as a closed circular molecule, but in some species (with large cpDNAs) a linear arrangement cannot be ruled out. The number of cpDNA molecules in a cell depends on two factors: the number of chloroplasts and the number of cpDNA molecules within each chloro

51、plast.,CHLOROPLAST DNA,All cpDNA molecules carry basically the same set of genes, but in different species these genes are arranged in different ways. The basic gene set includes genes for ribosomal RNAs, transfer RNAs, some ribosomal proteins, various polypeptide components of photosystems that are

52、 involved in capturing solar energy, four subunits of a ribulose 1,5-biphosphate carboxylase磷酸核酮糖梭化酶, and four subunit of a chloroplast-specific RNA polymerase. Most cpDNAs have a pair of large inverted repeats that contain the genes for ribosomal RNAs. These repeats range anywhere from 10 to 76 kb

53、in length and are variously located in different cpDNA molecules.,.,Genetic organization of the chloroplast DNA in the liverwort Marchantia polymorpha. Symbols: rpo, RNA polymerase; rps, ribosomal proteins of small subunit; rpl and secX, ribosomal proteins of large subunit; 4.5S, 5S, 16S, 23S, rRNAs

54、 of the indicated size; rbs, ribulose bisphosphate carboxylse; psa, photosystem I; psb, photosystem II; pet, cytochrome b/f complex; atp, ATP synthesis; infA, initiation factor A; frx, iron-sulfur proteins; ndh, putative NADH reductase; mph, chloroplast permease (?); tRNA genes are indicated by abbr

55、eviations for the amino acids.,CHLOROPLAST BIOGENESIS,All plastids develop from proplastids. Chloroplasts development is stimulated by light, and involves the transcription of many genes, some located in the nucleus. All the proteins and chlorophyll pigments needed for photosynthesis are made and ta

56、rgeted to their appropriate locations within the emerging chloroplast. The formations of functional chloroplasts is a process referred to as biogenesis. Only some of the details are known. Light plays an important role. Some genes are transcribed when light is provided.,CHLOROPLAST BIOGENESIS,A spec

57、ial class of pigmented proteins called phytochromes (光敏色素) seems to mediate this and others responses to light. By absorbing light energy, these proteins acquire the ability to trigger other proteins to stimulate the transcription of genes involved in chloroplast biogenesis. The formation of chlorop

58、lasts and the maintenance of their structure and function during the life of a plant depend on the coordinated expression of nuclear and chloroplast genes.,Chloroplast biogenesis. A mature chloroplast containing stacks (堆) of thylakoid類囊體 membranes (grana基粒) within its protoplasmic stroma (基質(zhì)) devel

59、ops from a proplastid after exposure to light.,KEY POINTS,Chloroplast DNA (cpDNA) molecules are typically 120 to 292 kb in size, and they contain at least 100 genes. The organization of cpDNA molecules varies among species of plants and algae. Light induces chloroplasts to develop from unpigmented plastids through a process that involves the interplay of chloroplast and nuclear gene products.,Mitochondrial Inheritance Yeast has been used extensively to study mitochondrial inheritance. There is a Yeast strain, called Petite小型菌聚落 that have structurally abnormal mitochondria that a