Organization of genes on chromosomes

• Mapping genes on chromosome:– Genetic mapping (linkage and recombination analys

es).

– Cytogenetics method.

– Restriction mapping, deletion studies, and other molecular approaches such as chromosome walking.

– Sequencing.

• Deduction of gene structure.

Restriction Mapping

Restriction Fragment Length Polymorphism (RFLP)

RFLP and VNTR (variable lengths of tandem repeats) areuseful in DNA fingerprinting.

Locating gene by chromosomal walking.

A case with the Duchenne muscular dystrophy gene.

Deduction of gene structure

• By hybridization of mRNA and chromosomal DNA.

• By restriction mapping.

• By DNA sequence.

Eukaryotic genes are often interrupted

Exons and introns

Organization of interrupted genes may be deduced byrestriction mapping of cDNA and genomic DNA

Small exons or introns may be missed by such an analysis. Resolution at the sequence level is necessary to identify allsegments of the gene.

Figure 4.12: Exons are identified by flanking sequences and ORFs.

Introns of nuclear genes generally have termination codons in all reading frames, and have no coding function.

Organization of interrupted genes may be conserved: all globin genes have the following interrupted structures.

Exon sequences are conserved but introns vary

Another example with the DHFR gene.

Frequency of interrupted genes in some eukaryotes

Exons are usually short

Some DNA sequences code for more than one protein

Two genes may share the same sequence by reading the DNAin different frames

Alternative splicing of mRNA can produce more than one protein

Genes can be identified and isolated by many approaches

• By classical genetic approach.• By reverse genetic approach. • DNA cloning and/or cDNA approach.• By conservation of exons (eg., zoo blotting).• By analysis of DNA sequence (ORF flanked by sp

licing junction). • By exon trapping.• By chromosome walking (especially for large-size

d gene).

How would you know that a segment of DNA is part of a gene?

1. Cross-hybridization with the genomes of other species (Zoo blot).2. Contains open reading frames (ORF).

Characterization of dystrophin gene by zoo blotting, cDNA identification and chromosomal walking.

Exon trapping

How did interrupted genes evolve?

Introns early or introns late?

1. The equation of at least some exons with protein domains, and the appearance of related exons in different proteins, indicates that the duplication and juxtaposition of exons has played an important role in evolution.

2. Most protein-coding genes probably originated in an interrupted form, but interrupted genes that code for RNA have originally been uninterrupted.

Every exon of immuoglobulin gene corresponds exactly with a know functional domain of the protein

Chromosomes, nucleosomes and controlling chromatin sturcture

• How is DNA packed in the chromosomes.

• Unusual chromosome structures.

• Nucleosomes.

• Controlling chromatin structure.

• Gene function and chromatin structure.

• Epigenetics (read Chapter 31 of Genes IX) .

DNA Topology

• Topology is a branch of mathematics that studies the properties of an object that do not change under continuous deformations. For circular DNA molecules, a topological property is one that is unaffected by deformations of the DNA strands as long as no breaks are introduced. DNA topology is the study of the spatial relationship of DNA.

• Topology of DNA– Intramolecular properties – relationship between the two strands of

the duplex. Only CCC DNA or linear duplex with at least two anchors are of topological concerns.

– Intermolecular properties – relationship between two molecules, eg., catenanes.

Supercoils

Supercoiling of DNA can only occur in closed-circular DNA or linear DNA where the ends are fixed.

Underwinding produces negative supercoils, whereas overwinding produces positive supercoils.

Negative and positive supercoils .

Topoisomerases catalyze changes in the linking number of DNA.

Relaxed and supercoiled plasmid DNAs

Topology of cccDNA is defined by: Lk = Tw + Wr, where Lk is the linking number, Tw is twist and Wr is writhe.

Intertwining of the two strands

• Nodes = ss crossing on 2D projection.

Right-handed crossing = +1/2

Left-handed crossing = -1/2

Lk = number of times one strand winds around the other on 2D projection.One linking number = 2 nodes.

DNA Compaction Requires Solenoidal Supercoiling, not plectonemic supercoiling.

Chromosome, chromatin, chromatid

• Chromatin: the complex of DNA and protein in the nucleus of the interphase cell. Heterochromatin refers to regions of the genome that are permanently in a highly condensed condition, while euchromatin refers to the rest of the genome.

• Chromosome: consists of one DNA molecule and proteins. Visible as morphological entity only during mitosis.

• Chromatids: copies of a chromosome produced by replication.

Packing ratio: the length of the DNA divided by the length of the unit that contains it.

Packaging of DNA

Bacterial chromosome

1. HU and H1 proteins may be involved in condensing DNA.2. There are about 400 domains of independent supercoiling, each consists of ~10-40 kb.3. The average density of

supercoiling is ~1 turn/100 bp.4. Treatment with reagents that act on RNA or protein may unfold the nucleoid. Question: How is #2-3 determined?

Loops, domains and scaffolds in eukaryotic DNA

• Genome, when isolated carefully, can be visualized as 10 nm fiber, consisting of DNA and protein. Supercoiling measured by EtBr indicates about 1 negative supercoils per 200 bp.

• Loops can be seen directly when the majority of histones are removed (see next Fig.). Threads of DNA emanate from the scaffold as loops of average length 10-30 m (30-90 kb).

Is DNA attached to the nuclear matrix or scaffold via specific sequences? Analysis of MAR(Matrix attachment region) does not reveal any conservation of sequence in MAR fragments. cis-acting sites that regulate transcription are common. A recognition site for topoII is usually present in the MAR.

Lampbrush chromosome

Polytene chromosomes

The length is the chromosome set is ~2000 m, whilethe DNA in extended form would stretch for ~40,000 m,so the packing ratio is about 20.

Centromeres:1. The sequences required for centromeric function has been identified in yeast S. cerevisiae (as shown below).2. The centromeres of S. pombe lie within regions of 40-100 kb that consist largely of repetitious DNA. The primary motif of primate centromeres is the satelllite.

Telomeres: consists of repetitive sequences rich in G.

SS G-tails in Telomere

Incomplete replication of lagging-strand at linear DNA ends.

Minimum features required for existence as a chromosome (YAC)

• Telomere to protect chromosome ends and to ensure survival.

• A centromere to support segregation.• An origin to initiate replication.

How is DNA packed in the chromosomes

• DNA supercoiling.

• Proteins assisted packaging (nucleosomes)

The nuclosome is the subunit of all chromatin

Packing ratio ~6.

DNA structure varies on the nucleosomal surface

The average periodicity of DNA in nucleosome is 10.2-10.4bp, which is slightly less than the 10.5 bp in free DNA.

Supercoiling and the periodicity of DNA: linking number paradox

SV40 DNA is 5.2 kb(1500 nm). In virionor infected nucleus, it is packaged into a seriesof nucleosomes with thecontour length of 210 nm. The no. of supercoilsmeasured approximates the no. of nucleosomes.

Path of nucleosomes in the chromatin fiber

Packing ratio ~40, suggesting ~6 nuclosomesfor every turn.

Model of DNA compaction in eukaryotic chromosomes

Organization of the histone octamer

Reproduction of chromatin requires assembly of nucleosomes

Histone octamers are not conserved during replication,Bit H2A-H2B dimers and H3-H4 tetramers are conserved.

Do nucleosomes lie at specific positions?

Position of DNA on nucleosomecan be important in controllingaccess to DNA. Displacement ofthe DNA by 10 bp changes the sequences in the linker regions,while a non-10 bp displacement can affect accessibility of DNA to different factors.

Are transcribed genes organized in nucleosomes?

The unifying model suppose thatRNA polymerase displaces histone octamers as it progresses. If the DNA behind the polymerase is available, the octamer reattches there. If the DNA is not available, eg., another polymerase continuesimmediately behind the first, thenthe DNA may persist in an extended form.

DNAase hypersensitive sites: usually found only in chromatin of cells in which the associated gene is being transcribed.

DNAase I sensitivity defines a chromosomal domain, a regionof altered structure including at least one active transcription unit.

29.14 Insulators Block the Actions of Enhancers and Heterochromatin

• Insulators are able to block passage of any activating or inactivating effects from:– Enhancers– Silencers– LCRs

Figure 29.42

• Insulators may provide barriers against the spread of heterochromatin.

Figure 29.43

29.15 Insulators Can Define a Domain• Insulators are specialized chromatin structures that have

hypersensitive sites.

• Two insulators can protect the region between them from all external effects.

Figure 29.44

29.20 An LCR May Control a Domain• An LCR (locus control region):

– is located at the 5′ end of the domain– consists of several hypersensitive sites

Figure 29.54

29.21 What Constitutes a Regulatory Domain?

• A domain may have:– an insulator– an LCR– a matrix attachment site– transcription unit(s)

Figure 29.54

Controlling Chromatin Structure

30.2 Chromatin Can Have Alternative States

• Chromatin structure:– is stable– cannot be changed by

altering the equilibrium of transcription factors and histones

Figure 30.2

30.3 Chromatin Remodeling Is an Active Process

• There are several chromatin remodeling complexes that use energy provided by hydrolysis of ATP.

Figure 30.3

• The SWI/SNF, RSC, and NURF complexes all:– are very large– they share some common subunits

• A remodeling complex does not itself have specificity for any particular target site.– It must be recruited by a component of the transcription apparatus.

Figure 30.5

30.4 Nucleosome Organization May Be Changed at the Promoter

• Remodeling complexes are recruited to promoters by sequence-specific activators.

• The factor may be released once the remodeling complex has bound.

Figure 30.6

• The MMTV promoter requires a change in rotational positioning of a nucleosome to allow an activator to bind to DNA on the nucleosome.

Figure 30.7

30.5 Histone Modification Is a Key Event

• Histones are modified by:– methylation– acetylation– phosphorylation

Figure 30.8

30.6 Histone Acetylation Occurs in Two Circumstances

• Histone acetylation occurs transiently at replication.

Figure 30.12

• Histone acetylation is associated with activation of gene expression.

Figure 30.13

30.7 Acetylases Are Associated with Activators

• Deacetylated chromatin may have a more condensed structure.

• Transcription activators are associated with histone acetylase activities in large complexes.

Figure 30.14

• Histone acetylases vary in their target specificity.

• Acetylation could affect transcription in a quantitative or qualitative way.

30.8 Deacetylases Are Associated with Repressors

• Deacetylation is associated with repression of gene activity.

• Deacetylases are present in complexes with repressor activity.

Figure 30.16

30.9 Methylation of Histones and DNA Is Connected

• Methylation of both DNA and histones is a feature of inactive chromatin.

• The two types of methylation event may be connected.

30.10 Chromatin States Are Interconverted by Modification

• Acetylation of histones is associated with gene activation.

• Methylation of DNA and of histones is associated with heterochromatin.

Figure 30.17

30.11 Promoter Activation Involves an Ordered Series of Events

• The remodeling complex may recruit the acetylating complex.

• Acetylation of histones may be the event that maintains the complex in the activated state.

Figure 30.18

30.12 Histone Phosphorylation Affects Chromatin Structure

• At least two histones are targets for phosphorylation, possibly with opposing effects.

30.13 Some Common Motifs Are Found in Proteins That Modify Chromatin

• The chromo domain is found in several chromatin proteins that have either activating or repressing effects on gene expression.

• The SET domain is part of the catalytic site of protein methyltransferases.

• The bromo domain:– is found in a variety of proteins that interact with chromatin

– is used to recognize acetylated sites on histones.