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Disease Genes

Chromosomal translocation analysis hybridization using banding patterns and multicolor FISH

Fluorescent in situ

(w. choromosome specific sequence probe)

Each chromosomes are stained with different color.

FISH can be used for molecular diagnostics

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Chromosomes

classes of DNA
Coding sequence (genes) Solitary genes vs Gene family Non-coding sequence Simple sequence repeats--highly repetitive Intermed. Repeats, moderately repeated mobile DNA insertion sequences transposons retrotransposons nonviral retrotransposons (SINEs, LINEs)

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Genes--Solitary or Gene family *gene duplication---unequal crossing over event ex. -like globin gene family

General Function---Carry oxygen in the blood A, G genes ---only expressed during fetal life, higher affinity for oxygen

Unequal cross over

*Tandemly repeated genes---rRNA, snRNA, histone genes *Pseudogenes

Comparisons of Related sequences from different species

Gene duplication and divergence give rise to protein families
Fig 9-32

Evolution

divergence

duplication

Orthologus Comparisons of sequences between

Homologous--Sequences are similar, common ancestral sequences Paralogous -tubulin from the different species

between -tubulin and -tubulin from the same species

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How do you find genes from the genomic sequences?

Bacteria, yeast ----relatively easy since no (or little) introns Identification of ORFs (open reading frames)--start and stop codons ~100 amino acids

How do you find genes from the genomic sequences ?

Higher eukaryotes---coding sequences (exons) are separated by introns Phylogenetic comparisons---Genes with related functions often have (1) similar amino acids, or (2) gene structures between different species.

consensus Sequences (common)

P-value--10-28

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Computer search for orthologs and paralogs

ORFs with similar amino acid sequences often have similar cellular functions. Among orthologs (or paralogs), amino acids may be similar while nucleotide sequences may not. ---Algorithms to translate the nucleotide sequences in three reading frames and search entire genome Genes (proteins) with similar functions sometimes have similar gene structures----exons & introns Specific protein domains---- exons

These information can also be used for finding potential functions of the unknown genes.

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classes of DNA
Coding sequence (genes) Solitary genes vs Gene family Non-coding sequence Simple sequence repeats--highly repetitive Intermed. Repeats, moderately repeated mobile DNA insertion sequences transposons retrotransposons nonviral retrotransposons (SINEs, LINEs)

Genome contains 1. Solitary genes vs Gene family 2. Pseudognes-----copies of genes that are nonfunctional even though they seem to have the same exon-intron structure as a functional gene Probably arise from a gene duplication followed by the accumulation of mutations that render the gene nonfunctional Non-coding sequence 3. Microsatellites----repeats of 1-13 bp, present in 25-50 repeat units within relatively short 1-5 kb regions 4. Minisatellites----repeats of 14-100 bp, present in 25-50 repeat units within relatively short 1-5 kb regions DNA fingerprinting---the number of copies of the tandemly repeated DNA sequences varies widely between individuals

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Mobile elements---Exon Shuffling, DNA fingerprinting, Pseudogenes, Processed pseudogenes

DNA intermediate

RNA intermediate

Transposition: process by which mobile sequence elements are copied and inserted into a new genomic site

5. Retrotransposons *viral transposons---LTRs (long terminal repeats) at their ends & reverse transcriptase and integrase * LINEs----no LTRs, instead containing direct repeats that flank a region that encodes RNA binding protein and reverse transcriptase *SINES----no LTRs, instead containing direct repeas, no ORFs 6. DNA transposons * bacteria----IS (insertion sequence) element, usually contains inverted repeats that flank transposase Insertion of transposons within the genome can generate spontaneous mutations, or unequal crossing over between them at different chromosomal locations leads to exon duplications, gene duplications, and chromosomal rearrangements

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Mammalian retroposons 1. Viral Superfamily 2. LINEs 3. SINEs 1. Viral Superfamily Ex. Ty (S.cerevisiae), copia (Dros) Termini----------Long terminal repeats (LTRs) Target repeats----4-15 bp Enzyme activities---Reverse transcriptase, integrase

2. LINES

Example. L1 (Human)

Termini----------No repeats Target repeats----7-21 bp Enzyme activities---Reverse transcriptase, endonuclease

3. SINES

Alu sequence

no ORFs

Termini----------No repeats Target repeats----7-21 bp Enzyme activities---no coding for transposon products

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Experiment to show that the yeast Ty element transposes through an RNA intermediate

Growth of yeast cells in Galactose increased frequencies of transposition

Transposition into RNA is required for Ty transposition Ty element involves an mRNA intermediate.

Unequal Crossing Over

*Tandemly repeated genes---rRNA, snRNA, histone genes

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Evolution and expansion of the genomic repertoire by mobile DNA elements

SINEs---Alu elements, very common in the human genome

Exon shuffling via recombination between Alu sequences

Mobile DNA elements could serve as recombination sites to mobilize adjacent DNA sequences

Exon Shuffling

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Simple Sequence Repeats--highly repetitive

Non-coding DNA *Short repeats---5-10 bp *Satellite DNA-----relatively short, nearly identical repeats ~3% of the human genome 14-500 bp in tandemly repeats of 20-100 Kb The length of a particular simple sequence tandem array is variable between individuals in a species

Human diseases caused by microsatellites neuromuscular diseases some are recessive but other behave like dominant DMPK gene----contains 100-4000 CUG repeats (normally 50-100 CUG repeats) in the 3 UTR. The extended CUG repeats affect mRNA processing and export of mRNA.

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Human metaphase chromosomes Minisatellite DNA used for DNA finger printing Fluoresce in situ hybridization 1-5 kb regions, scattered 20-50 repeat units of 15-100 bp (FISH) sequence

Southern blot or PCR

Restriction Enzyme digest of genomic DNA, Southern blot

Simple-sequence DNA Repeats are found in Centromeres, telomeres

unit repeat conserved but # of repeats varies

..can be used in fingerprinting

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