FISH analysis of chromosomes: Fluorescent IN SITU hybridization

metaphase spread chromosomes stained with DAPI, a fluorescing stain that specifically binds double stranded DNA

Expose DAPI-stained metaphase chromosomes to fluorescent probes red = control probe for centromere of the X chromosome & another probe for end of chromosome X

green = probe for end of chromosome 4

DiGeorge syndrome/CATCH22
microdeletion on chromosome 22 birth defect that affects the immune system absence of or underdevelopment of the thymus and parathyroid glands facial features include low-set ears, wide-set eyes, small jaw, and bowing up of upper lip

FISH tests: DiGeorge syndrome

Expose DAPI-stained chromosomes to mixture of fluorescent probes green = control probe for chromosome 22 red = probe for DiGeorge region on long arm of chromosome 22

FISH tests: Painting chromosomes

Expose chromosomes to fluorescent probes that highlight entire chromosomes

FISH tests: Painting chromosomes

Expose chromosomes to fluorescent probes that highlight chromosomes 13, 18, 21, X, and Y

nuclei from the same fetus

green = chromosome 13 red = chromosome 21

aqua = chromosome 18 green = X chromosome red = Y chromosome

DNA Recombination
General recombination Site specific recombination

General DNA Recombination

Heteroduplex joint

General Recombination
Two homologous DNA molecules cross over The site of exchange can occur anywhere A strand of one DNA molecule has become base-paired to a strand of the second DNA to create heteroduplex joint No nucleotide sequences are altered

The procedure of general recombination

DNA synapsis: base pairs form between complementary strands from the two DNA molecules

DNA Hybridization The initial step for DNA recombination

RecA protein-mediated DNA synapsis Rec A has multiple DNA binding sites, hence can hold a single strand and a double helix together Rec A is also a DNA-dependent ATPase

DNA Branch Migration

Holiday Junction for DNA recombination Exchange of the first single strand between two different DNA double helices is slow and difficult, then intermediate state Holiday Junction, then complete exchange

Holiday Junction for DNA recombination and its resolution

Summary for General Recombination

General recombination allows large fraction of genetic information to move from one chromosome to another. General recombination requires the breakage of double helices, beginning with a single strand breakage. General recombination is facilitated by Rec A in bacteria and its homologs in eucaryotes. Holiday junction is the intermediate state of general recombination

Site-specific recombination
Moves specialized nucleotide sequence (mobile genetic elements) between non-homologous sites within a genome. Transpositional site-specific recombination Conservative site-specific recombinatinon

Transpositional site-specific recombination

Modest target site selectivity and insert mobile genetic elements into many sites Transposase enzyme cuts out mobile genetic elements and insert them into specific sites.

Three of the many types of mobile genetic elements found in bacteria Transposase gene: encoding enzymes for DNA breakage and joining Red segments: DNA sequences as recognition sites for enzymes Yellow segments: antibiotic genes

Cut and Paste Transposition DNA-only

The structure of the central intermediate formed by transposase (integrase)

Replicative Transposition

Retrovirus-based Transposition Retroviral-like retrotransposition

Reverse Transcriptase

RNA

Non-retroviral retrotransposition

Conservative Site Specific Recombination Integration vs. inversion Notice the arrows of directions