Transcript DNA、RNA、蛋白質、糖類

分子診斷學概論
 第一章 綜說 overview
疾病發生原因的影響層次
DNA、RNA或蛋白質
分子診斷的目的
偵測這些致病因子是那個層次發生變化
本書著重DNA、RNA的變化
蛋白質層次由原文書章節提供
The Application of Proteomics To
Disease Diagnostics
 遺傳分子的基礎
生物巨分子：DNA、RNA、蛋白質、糖類、脂
質
遺傳物質DNA的發現
1928 格里夫茲 (Griffith)肺炎雙球菌轉形試
驗
1942 艾佛瑞(Avery)研究格里夫茲轉形的物
質為何?
1952 赫希-卻斯 (Hershey-Chase)以放射線
標示噬菌體的蛋白質（S35）和DNA（P32），
感染大腸桿菌的實驗
1953 雙螺旋結構的發現
2003 人類基因體計畫的完成
參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg
參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg
www.accessexcellence.org
參考資料：http://biotech.nstm.gov.tw/advance/a021.asp
 Presented here is a genome sequence of an individual
human. It was produced from ~32 million random DNA
fragments, sequenced by Sanger dideoxy technology
and assembled into 4,528 scaffolds, comprising 2,810
million bases (Mb) of contiguous sequence with
approximately 7.5-fold coverage for any given region.
We developed a modified version of the Celera
assembler to facilitate the identification and comparison
of alternate alleles within this individual diploid genome.
Comparison of this genome and the National Center for
Biotechnology Information human reference assembly
revealed
 more than 4.1 million DNA variants, encompassing 12.3 Mb.
These variants (of which 1,288,319 were novel) included
3,213,401 single nucleotide polymorphisms (SNPs), 53,823
block substitutions (2–206 bp), 292,102 heterozygous
insertion/deletion events (indels)(1–571 bp), 559,473
homozygous indels (1–82,711 bp), 90 inversions, as well as
numerous segmental duplications and copy number
variation regions. Non-SNP DNA variation accounts for 22%
of all events identified in the donor, however they involve
74% of all variant bases. This suggests an important role for
non-SNP genetic alterations in defining the diploid genome
structure. Moreover, 44% of genes were heterozygous for
one or more variants. Using a novel haplotype assembly
strategy, we were able to span 1.5 Gb of genome sequence
in segments .200 kb, providing further precision to the
diploid nature of the genome. These data depict a definitive
molecular portrait of a diploid human genome that provides
a starting point for future genome comparisons and enables
an era of individualized genomic information.
 Author Summary
 We have generated an independently assembled diploid human
genomic DNA sequence from both chromosomes of a single individual
(J. Craig Venter). Our approach, based on whole-genome shotgun
sequencing and using enhanced genome assembly strategies and
software, generated an assembled genome over half of which is
represented in large diploid segments (.200 kilobases), enabling study
of the diploid genome. Comparison with previous reference human
genome sequences, which were composites comprising multiple
humans, revealed that the majority of genomic alterations are the wellstudied class of variants based on single nucleotides (SNPs). However,
the results also reveal that lesserstudied genomic variants, insertions
and deletions, while comprising a minority (22%) of genomic variation
events, actually account for almost 74% of variant nucleotides.
Inclusion of insertion and deletion genetic variation into our estimates
of interchromosomal difference reveals that only 99.5% similarity
exists between the two chromosomal copies of an
individual and that genetic variation between two
individuals is as much as five times higher than previously
estimated. The existence of a well-characterized diploid human
genome sequence provides a starting point for future individual
genome comparisons and enables the emerging era of individualized
genomic information.
Identification and analysis of functional elements in 1%
of the human genome by the ENCODE pilot project.
Nature. 2007 Jun 14;447(7146):799-816
 The Encyclopedia of DNA Elements (ENCODE) Project
provide a more biologically informative
representation of the human genome by using
high-throughput methods to identify and catalogue
the functional elements encoded.
 First, our studies provide convincing evidence that the
genome is pervasively transcribed, such that the
majority of its bases can be found in primary transcripts,
including non-protein-coding transcripts, and those that
extensively overlap one another. Second, systematic
examination of transcriptional regulation has yielded
new understanding about transcription start sites,
including their relationship to specific regulatory
sequences and features of chromatin accessibility and
histone modification. Third, a more sophisticated view
of chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional
regulation. Finally, integration of these new sources of
information, in particular with respect to mammalian
evolution based on inter- and intra-species sequence
comparisons, has yielded new mechanistic and
evolutionary insights concerning the functional
landscape of the human genome. Together, these
studies are defining a path for pursuit of a more
comprehensive characterization of human genome
function.
 The highlights of our findings to date include
The human genome is pervasively transcribed,
such that the majority of its bases are associated
with at least one primary transcript and many
transcripts link distal regions to established
protein-coding loci.
Many novel non-protein-coding transcripts have
been identified, with many of these overlapping
protein-coding loci and others located in regions
of the genome previously thought to be
transcriptionally silent.
Numerous previously unrecognized transcription
start sites have been identified, many of which
show chromatin structure and sequence-specific
protein-binding properties similar to wellunderstood promoters.
Regulatory sequences that surround transcription
start sites are symmetrically distributed, with no
bias towards upstream regions.
Chromatin accessibility and histone modification
patterns are highly predictive of both the
presence and activity of transcription start sites.
Distal DNaseI hypersensitive sites have
characteristic histone modification patterns that
reliably distinguish them from promoters; some of
these distal sites show marks consistent with
insulator function.
DNA replication timing is correlated with
chromatin structure.
A total of 5% of the bases in the genome can be
confidently identified as being under evolutionary
constraint in mammals; for approximately 60% of
these constrained bases, there is evidence of
function on the basis of the results of the
experimental assays performed to date.
Although there is general overlap between
genomic regions identified as functional by
experimental
assays
and
those
under
evolutionary constraint, not all bases within these
experimentally defined regions show evidence of
constraint.
Different functional elements vary greatly in their
sequence variability across the human population
and in their likelihood of residing within a
structurally variable region of the genome.
Surprisingly, many functional elements are
seemingly unconstrained across mammalian
evolution. This suggests the possibility of a large
pool of neutral elements that are biochemically
active but provide no specific benefit to the
organism. This pool may serve as a 'warehouse'
for natural selection, potentially acting as the
source
of
lineage-specific
elements
and
functionally conserved but non-orthologous
elements between species
DNA的組成
H
(王文姿等，2003)
遺傳物質：核酸(nucleic acid)
核酸：DNA（去氧核醣核酸）、RNA（核醣核酸）
核酸基本單位：核苷酸(nucleotide)
核苷酸：鹼基(base)、五碳醣(pentose sugar)、磷酸(phosphate)
DNA
去氧核糖核酸
H
RNA
核糖核酸
鹼基
DNA的結構
主溝 major groove
小溝 minor groove
資料來源：http://academic.brooklyn.cuny.edu/biology
DNA的結構
二個氫鍵
穩定力量來自
氫鍵
非共價鍵-堆積
力量：凡得瓦力、
斥水性作用力、
親水性作用力
糖骨架：磷酸
雙酯鍵
三個氫鍵
三個氫鍵
二個氫鍵
資料來源：http://academic.brooklyn.cuny.edu/biology
環境與序列的影響會形成不同形式結構的DNA
較寬與緊密
外表成鋸齒狀
http://nucleix.mbu.iisc.ernet.in/image/abzDNA.JPG
基因的一般結構
基因定義：染色體上一段有功能的特定序列，
可轉錄成RNA分子或是轉譯成多胜肽鏈
定義可能會更改：調控性功能的序列？
 基因的結構
表現子（exon外顯子）-可轉錄或轉譯出產物，稱為編
碼區
轉錄調控區
啟動子promoter：其特殊序列與轉錄因子結合，引
導RNA聚合酶，產生基因轉錄
轉錄起始點定為+1
轉錄進行的方向稱為下游
相反的方向稱為上游
保留性序列
TATA box大約在-10 ~ -35處
CAAT box大約在 -75處影響啟動子效率
GC box由GGGCGG序列組成，也稱為sp1 box，
為轉錄因子sp1結合位置
Sp 1分離自人類細胞
由加州大學柏克萊分校的Tjian教授的實
驗室分離
可有效地促進猿猴濾過性病毒（simian
virus 40﹐簡稱SV40）基因的轉錄
加強子Enhancer
加強特殊基因的轉錄活性
位置不固定
與調節蛋白結合，與啟動子間形成圈環構造
靜默子silencer
與增強子功能相反的調控
反應元response element
與轉錄因子結合，作用到啟動子，增強轉錄
作用。
與特異性刺激因子結合，調節基因表現
TRE-TPA response element, SRE-serum
response element, HSE-heat shock response
element, CRE-Camp response element, EREestrogen response element, GREglucocorticoid response element, MRE-metal
response element….
阻絕子insulator
一段0.5 ~ 3 kb的DNA，阻斷轉錄因子散佈
（可能增強子或靜默子）。
 轉錄區
表現子exon（外顯子）
具有轉譯成多胜肽鏈或轉錄成RNA分子的序
列，為編碼區
內含子intro（插入子）
與exon一起被轉錄，但在mRNA剪切時會
被切除。5端 GT-AG 3端
可能與調控有關，基因的穩定有相關
聚腺苷酸化訊號polyadenylation signal
mRNA 3端的AAUAAA序列下游約15-30 bp
會被切除，再加上poly A tail。
 遺傳中心法則
DNA→RNA→蛋白質
mRNA的剪切由核內的小分子RNA (snRNA)
和snRNA蛋白質複合體(snRNPs)及SR蛋白
質參與，這些分子形成剪接體結構
(splicesome)
反轉錄酶的發現顛覆了此中心法則
1970 Howard Termin和David Baltimore
發現 (1975 Nobel)
一些RNA病毒能利用反轉錄酶將它們的遺傳
物質(RNA)反轉錄為DNA