Gene Structure and Function ZHANG Xian-Ning, PhD zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: C303, Teaching Building 2015/09

Cells = 2 organisms:nucleus – cytosol + mt. An adult organism has an estimated 1014 cells. Cell types: >200 Cells = 2 organisms:nucleus – cytosol + mt.

Human Genome Total genetic information in a living organism. Total genes in a haploidy set of chromosomes. Human genome: 3.1×109 bp Enough for encoding:1.5×106 proteins

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The Central Dogma

The RNA transcript is complementary to the template strand of the DNA and has the same 5'->3' direction and base sequence (except that U replaces T) as the opposite, nontemplate DNA strand. The nontemplate strand is often called the sense strand, and the template strand is often called the antisense strand.

Gene: a basic unit of heredity Chemically: A specific DNA fragment, which can be duplicated and mutated. In physics: Arranged on chromosome linearly, can be exchanged and transmitted to the next generation. Functionally: Controlling the expression of specific characteristics of a living organism.

Structural genes Genes directing the synthesizing proteins Only about ~ 20 000 – 25 000 pr.-coding genes (~ 20 000 – 25 000 noncoding RNA genes) 1.5%~2% of genome Normally in non-repetitive or low repetitive DNA

The regions of non-structural genes Spacer sequences: Non-encoding region, probably, regulatory genes for DNA duplication, transcription and mRNA translation Others: Insertion sequence, highly repetitive sequence, inverted repetitive sequences and sporadic moderately repetitive and low-repetitive DNA

Gene structure Gene is splited, alternate with the exons and the introns. Gene can be expanded, including the regulatory regions on both sides which are required for initiating and terminating gene expression sometimes.

Split Gene Structural genes in eukaryote are split genes with two kinds of sequences, although it is continuous in prokaryote. Exon: Encoding sequences, corresponding to the sequence of mRNA Intron (IVS): Non-encoding sequences, corresponding RNA sequence will be removed from mRNA.

Gene Structure：split gene

Functions of exon and intron Exons are encoding sequences and their mutations will influence the formation of proteins. Introns are non-encoding sequences and their mutations do not affect the protein formation and structures normally. However, sometimes, the mutation may influence the splicing of RNA and affect the production of mRNA

The number of exons and introns is variable Transcription starts and terminates on the exons, therefore, the numbers of exons and introns in a gene are N and N -1 respectively. The numbers of exons and introns in a gene is significantly variable: Beta-hemoglobin gene: 3 exons, 2 introns, 1 700 bp Duchenne Muscular Dystrophy gene: 79 exons, ~ 2 300 000 bp

β-globin gene

Flanking sequence Gene can be expanded, including the regulatory regions on both sides which are required for initiating and terminating gene expression sometimes: Promoter, Enhancer, Terminator

Splice site In the junction region of intron and exon 5’ intron: splice donor site 3’ intron: splice acceptor site. Either of which exists the same consensus sequences: Exon…AG↓GTAAGT… Intron… Py10CAG↓…Exon, and that is RNA splice codes Intron always sarts at GT … and ends at …AG: GT-AG Rule

Nomenclature of genes & proteins http://www.gene.ucl.ac.uk/nomenclature/index.html Antonarakis SE.  (1998) Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat. 11:1-3. den Dunnen JT, Antonarakis SE. (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion. Hum Mutat. 15:7-12.

Mutation Nomenclature Examples c.1444g>a: a mutation at position 1444 in the hexosaminidase A cDNA causing Tay-Sachs disease g.IVS33+2T>A: a mutation substituting an A for T in a splice donor site GT of intron 33 of a gene g.IVS33-2A>T: a mutation substituting a T for an A in the highly conserved AG splice acceptor site in the same intron c.1524_1527delCGTA: a deletion of four nucleotides, numbers 1524 through 1527 in cDNA c.1277_1278insTATC: a four-base insertion between nucleotides 1277 and 1278 in the hexosaminidase A cDNA, a common mutation causing Tay-Sachs disease p.Glu6Val: a missense mutation, glutamic acid to valine at residue 6 in β-globin, that causes sickle cell disease p.Gln39X: a nonsense mutation, glutamine to stop codon (X) at position 39 in β-globin, that causes β0-thalassemia

Transcription begins near promoter sites and ends at terminator sites A Promoter is a region of DNA where RNA polymerase binds to initiate transcription.

TATA box Hogness box Upstream of transcription start site: -19 ~ -27 bp Composed of TATATAT，highly conserved Identify the start site of transcription after combining with transcript factor TFII and RNA polymerase II.

CAAT box Upstream of transcription start site: -70 ~ -80 bp Composed of GGGTCAATCA Recognized by transcript factor CIF and promoting transcription. Mutation: Reduction of transcription levels

GC box Two copies between the two sides of CAAT box Composed of GGCGGG Combined by transcript factor SP1 and promoting transcription.

Enhancer A DNA sequence enhancing the promoter function Located in either upstream or downstream of promoter or transcription start site, 3kb or more Both 5’-3’ or 3’-5’ directions Function on both the genes in upstream and downstream.

Enhancer Promoter-specific: only enhancing the transcription of gene in which the promoter is corresponding with the enhancer. Tissue-specific: Immunoglobulin gene enhancer is only functioned actively in B lymphocyte

Terminator The end of downstream of gene Terminate the transcription of RNA Composed of a reversed repetitive sequence and specific 5′-AATAAA-3′. Reversed repetitive sequence: terminating signal AATAAA: Coding for polyA tail.

Different genes can share the same DNA sequence Same DNA sequence can encode different proteins

Exons and introns can be changed

Gene clusters Human hemoglobins

More concepts Some DNA sequences code for more than one protein Gene family or gene clusters Transcription factors Gene splicing

Transcription factors (TFs) Response elements: regulated in response to certain TFs Each TF has a specific binding sequence TFs have primary responsibility for recognizing promoter, binding to the RNA pol and positioning it correctly at the startpoint cis-acting elements in promoter Formation of pre-initiation complex

The regulatory region of human metallothionein gene

Splicing Occurs in the nucleus, together with other modifications (5’Cap, 3’Poly A) Three major mechanisms to remove introns Spliceosome Catalytic RNA Enzymatic removal (yeast tRNA)

Gene Structure and Expression

Human Gene Intron Structure

Mutations A mutation is a structural change in genomic DNA sequence due to errors in DNA replication or repair. Mutations may or may not result in an expressed phenotype. Mutations that have no phenotype are called neutral mutations. - Mutations can alter RNA expression, processing and/or stability. - Mutations can also affect protein expression, processing, stability. Mutations can be inherited (genetic/germline mutations) not inherited (somatic mutations)

Structural classification of mutations 1. Point mutations: change in one base pair of DNA. (1) silent mutations: changes in DNA which do not affect protein expression or function. (2) missense mutations: changes in DNA which lead to a change in an amino acid. (3) nonsense mutations: changes in DNA which generate a termination codon and thus stop translation. (4) Regulatory mutations: one which involves the promoter or another regulatory sequence such as an enhancer, silencer, or locus control region. (5) RNA processing mutations: These affect the processing of the primary RNA transcript to form mRNA, either by altering normal RNA splicing or by preventing either normal 5’-capping or 3’-polyadenylation.

Types of Mutations Silent mutation Missense mutation Non-sense mutation Frameshift mutation

Structural classification of mutations 2. Deletions and insertions: small deletions and insertions: If the number of nucleotides deleted or inserted in an exon is not a multiple of three, then the sequence of codons, known as the reading frame, is disrupted. This is referred to as a frame-shift → a truncated protein. large deletions and insertions: These range in size from 20 bp to 10 Mb, beyond which they become visible using a light microscope and are classified as chromosome abnormalities. unequal crossing-over: Crossing-over between misaligned closely adjacent sequences which show close homology results in the formation of a deletion in one chromatid and a duplication in the other. (4) retrotransposition: Transposable elements, SINES and LINES, which have moved from an inert region of the genome to become inserted into an exon elsewhere.

Polymorphisms ‘Mutations’ that are propagated and maintained in the population at relatively high frequencies are called polymorphisms. Polymorphism is defined as the existence of two or more alleles, where the rare allele appears with a frequency greater than 1% in the population. Most mutations are quickly lost from population due to deleterious effects (natural selection) or genetic drift (random fluctuations). Mutations may become polymorphisms due to selective advantage (heterozygotes for hemoglobin sickle cell mutation are more resistant to malaria) or genetic drift (founder effect, small group of individuals found a new population).

Polymorphism Multiple functional alleles at a frequency of >1% in a population A population may have extensive polymorphism at the level of genotype It may offer a diagnostic procedure for detecting the disease It may lead to isolation of the gene

SNP（单核苷酸多态性）: A change in which a single base in the DNA differs from the usual base at that position. SNPs are the most common type of variations. Millions of SNP's have been cataloged in the human genome. Some SNPs such that which causes sickle cell are responsible for disease. Other SNPs are normal variations in the genome.

Copy-number variations (CNVs): a form of structural variation - are alterations of the DNA of a genome that results in the cell having an abnormal number of copies of one or more sections of the DNA.

“You are only one and they are so many “You are only one and they are so many. You must work day and night, or else you will be crushed like a roach!” ----Gunter Blobel (1999)