Course 72332, mobile DNA : Evolutionary changes in genetic information Pages to read: Lodish (Ch. 10.3),

What is a genome? ► "DNA makes RNA, RNA makes protein, and proteins make us." Francis Crick ► Gene is a functional chromosomal DNA area which comprises of promoter, transcription start site (TSS) and coding sequence.

Indian Corn Barbara McClintock

Mobile DNA ► Non coding repetitious DNA constitutes a significant fraction of genomic DNA in higher eukaryotes ► Moderately repeated DNA sequences are mobile ► About 100 – 7000 base pairs in length ► Present in both Eukaryotes and prokaryotes ► Copied and inserted into a new site in the genome by Transposition.

Mobile DNA elements are molecular parasites ► As parasites, they do not participate in the functions of the host cell ► They exist to maintain themselves : ”selfish DNA” [ F. Crick ] ► They slowly accumulate in eukaryotic genomes over evolutionary time. ► They also accumulate mutations and sometimes get deleted and eliminated

Movement of Mobile elements involves a DNA or RNA intermediate ► Barbara McClintock – corn variants are unstable due to mobile elements moving in and out [ 1940 ] ► Bacterial mobile elements show similarity to eukaryotic intermediate repeats [ 1990 ] ► Mobile elements may transpose directly or through an RNA intermediate ► The latter involve transcription of RNA, reverse transcription into DNA and double strand formation ► Therefore, they are called retrotransposons

Classification of mobile elements into two major classes Retrotransposons move Analogously to the infectious process the infectious process Of retroviruses Retroviruses can be regarded as regarded asretrotransposons that evolved into a virus that evolved into a virus with coat proteins that with coat proteins that facilitate Transposition facilitate Transposition between cells between cells

Mobile elements that move as DNA ► Present in both prokaryotes and eukaryotes ► Most mobile elements in eukaryotes are Retrotransposons ► The famous corn transposons, however, move as DNA ► E. coli IS elements are 1-2 kb long and form heteroduplexes with their variants ► IS transposition occurs once in 100, million cells per generation

General structure of bacterial IS elements Encodes for enzymes Enabling transposition Direct repeats are Generated from target site DNA during insertion

The biological significance of transposition in eukaryotes ► High rates will endanger the survival of the overly mutated cell ► Low rates will ascertain propagation of the mobile element ► Many transpositions inactivate essential genes, killing the host and the element ► Transposition is random and often involves non-essential DNA regions ► In such cases, it will expand the mobile DNA

Mobile DNA insertion into non-cellular sites ► Insertion can occur in plasmids or lysogenic viruses ► These can transform into cells ► The result is IS element transposition into virgin cells ► Expression is very slow, hence the slow transposition rate

Antibiotics Resistance Antibiotics Resistance

There are various mechanisms for antibiotic resistance

Tuberculosis Notification Rates, 2000

► anti.m1v anti.m1v

The corresponding processes

General Structure of bacterial transposons Include IS elements flanking coding sequences that encode antibiotics resistance genes that encode antibiotics resistance genes Explain acquired resistance of bacteria To antibiotics Provide a valuable tool for research And a selection marker

. Three of the many types of mobile genetic elements found in bacteria ► Two of the three mobile elements carry genes that encode enzymes that inactivate the antibiotics ampicillin (ampR) and tetracycline(tetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two short mobile elements on either side of a tetracyclin- resistance gene; the wide use of tetracycline as an antibiotic has aided the spread of this gene through bacterial populations. The three mobile elements shown are all examples of DNA-only transposons. genesenzymesantibioticstransposable elementgeneantibioticgeneDNA-only transposonsgenesenzymesantibioticstransposable elementgeneantibioticgeneDNA-only transposons

Model for nonreplicative transposition of bacterial insertion sequences of bacterial insertion sequences Relevant enzymes: Transposase DNA polymerase Ligase The single copy Element is transferred as is to a new location to a new location In other cases, the element is replicated And appears in Two locations

. Cut-and-paste transposition ► DNA-only transposons include “inverted repeat DNA sequences” (red) at their ends. These sequences can be as short as 20 nucleotides, and are all that is necessary for the DNA between them to be transposed by the particular transposase enzyme associated with the element. The cut-and-paste movement begins when the transposase brings the two inverted DNA sequences together, forming a DNA loop. Insertion into the target chromosome, catalyzed by the transposase, occurs at a random site through the creation of staggered breaks in the target chromosome(red arrowheads). The insertion site is marked by a short direct repeat of the target DNA sequence. The break in the donor chromosome (green) is resealed, and the breakage-and-repair process often causes a mutation at the original site of the excised transposable element. DNA-only transposonsnucleotidesenzymechromosome mutationtransposable element DNA-only transposonsnucleotidesenzymechromosome mutationtransposable element

The structure of the central intermediate formed by a cut-and-paste transposase The structure of the central intermediate formed by a cut-and-paste transposase (A) Schematic view of the overall structure. (B) The detailed structure of a transposase holding the two DNA ends, whose 3′-OH groups are poised to attack a target chromosome. chromosome

McClintock’s corn included two types ► Mutations affect anthocyanine production ► Mutants are white, WT – purple ► Activator mutations [Ac ] are reversible at high frequency ► Dissociation mutations [ Ds ] do not revert unless Ac mutation is present ► Ds mutations associate with chromosome breaks ► Ds elements are deleted forms of Ac elements ► Deletions impair the transposase, so movement is prevented

Transposons occur in many species ► 50% of spontaneous mutations in Drosophila ► Most are retrotransposons, but P elements are transposons ► P element transposase serves to engineer flies ► Retrotransposons divide into viral and non-viral ► Yeast Ty elements and Drosophila copia elements are viral retrotransposons ► 4% of human DNA is viral retrotransposons

General structure of eukaryotic viral of eukaryotic viral retrotransposons retrotransposons Long terminal repeats [ LTR ] of viral origin May represent descendents of viral infections Or a prototypic oncovirus without coat protein Encode proteins of retroviruses Which enable expression of the retrotransposon

. The life cycle of a retrovirus ► The retrovirus genome consists of an RNA molecule of about 8500 nucleotides; two such molecules are packaged into each viral particle. The reverse transcriptase first makes a DNA copy of the viral RNA and then a second DNA strand, generating a double-stranded DNA copy of the RNA genome. The integration of this DNA double helix into the host chromosome is then catalyzed by a virus-encoded integrase. This is required for the synthesis of new viral RNA molecules by the host cell RNA polymerase, the enzyme that transcribes DNA into RNA. retrovirusgenomemoleculenucleotidesmoleculesreverse transcriptasegenomedouble helixchromosomevirusmoleculesRNA polymeraseenzymeretrovirusgenomemoleculenucleotidesmoleculesreverse transcriptasegenomedouble helixchromosomevirusmoleculesRNA polymeraseenzyme

Generation of retroviral genomic RNA from integrated retroviral DNA Promoter

Retrotransposons use viral and cellular enzymes ► Cellular RNA polymerase II copies the sequence,starting at left LTR ► Right LTR directs cellular enzymes to cleave the transcript and add poly [A] tail ► Viral reverse transcriptase forms double strand DNA with LTRs ► Viral integrase inserts the DNA into the host cell genome ► Short direct repeats are formed from integration site sequence

Generation of LTRs during reverse transcription of retroviral genomic RNA Retrovirus tRNA Is packaged within The virion tRNAhybridizes With primer Binding site [ PBS ]

R=short direct repeat repeat Generation of LTRs during reverse transcription of retroviral genomic RNA (cont.)

Generation of retroviral genomic RNA from integrated retroviral DNA ► Short direct repeats of target site DNA are generated during integration of the retroviral DNA into the host cell genome ► The left LTR directs cellular RNA polymerase II to initiate transcription at the 1 st nucleotide of the left R region. ► The primary transcript extends beyond the right LTR. ► Cellular enzymes cleave the RNA at the end of the right R region ► Polyadenylation yields retroviral RNA with a poly (A) tail

Ty elements transpose through an RNA intermediate ► Yeast genome includes little spacer and intron DNA ► Therefore, transposition is likely to cause lethality and occurs in a slow rate ► Ty plasmids with galactose –sensitive promoter increase Ty transposition in the presence of galactose ► Inserted intron-an RNA sequence that is cleaved and does not end up in the mRNA- did not appear in transposed Ty elements, suggesting splicing ► In contrast, Ac elements include introns, as they insert directly

Experimental demonstration that yeast Ty element moves through an RNA intermediate

Nonviral retrotransposons lack LTRs and move by an Unusual mechanism ► Long interspersed elements- LINES ; 6-7 kb ► Short interspersed elements- SINES ; 300 bp ► Thousands copies each, not necessarily exact ► Especially abundant in mammals ► Likely transpose through intermediate RNA

. Transpositional site-specific recombination by a non-retroviral retrotransposon TranspositionTransposition by the L1 element (red) begins when an endonuclease attached to the L1reverse transcriptase and the L1 RNA (blue) makes a nick in the target DNA at the point at which insertion will occur. This cleavage releases a 3′-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown. This generates a single-stranded DNA copy of the element that is directly linked to the target DNA. In subsequent reactions, not yet understood in detail, further processing of the single- stranded DNA copy results in the generation of a new double-stranded DNA copy of the L1 element that is inserted at the site where the initial nick was made. reverse transcriptasecleavagereactions Transpositionreverse transcriptasecleavagereactions

General structure of an L1 LINE element, A common type of eukaryotic nonviral retrotransposon 600,000 copies in the human genome – 15% of our DNA (!) ORF1 encodes an RNA-binding protein ORF2 encodes a retroviral reverse transcriptase

The mobility of L1 elements in the human genome ► Novel insertion into genes of carriers of inherited diseases ► Genetic engineering (intron insertion) proof that RNA intermediate was involved, since splicing occurred ► But L1 elements don’t have LTRs, hence a different mechanism must be involved

Proposed mechanism of nonviral retrotransposition of L1 elements of L1 elements Most L1 sequences Accumulated stop Codons in ORF1 and ORF2. Therefore, mobilization is Extremely slow One L1 sequence With intact ORFs Can induce transposition Of others

SINES and Alu sequences ► 2 nd major class of moderately repeated DNA in mammals,1 million copies in humans (10% of our DNA ), plus many fragments of them ► None are identical, but they show similarity to Ty elements in yeast ► 80% conserved within species, % between species ► Often include Alu1 restriction sites ► Similar to 7SL RNA, participating in secretion processes in all organisms –even E.coli ► Missing in Drosophila or yeast, arose later in evolution from 7SL RNA

Alu and neurofibromatosis ► NF1 mutations cause multiple neuronal tumors – neurofibromas ► Develop only when both alleles are mutated ► Alu transposition into the NF1 gene was shown to cause neurofibromatosis in a patient ► Disease developed following somatic mutations in the other allele Neuroma tumors generated by the insertion of a SINE element into the NF1 gene

Alu expression ► Transcribed by RNA polymerase III,do not encode proteins ► Include an A/T rich sequence at one end, like L1 elements ► Believed to be retrotransposed by reverse transcriptase of L1 elements ► Appear between genes, within introns and transcribed non – coding domains of many genes ► Have no known function

Processed pseudogenes ► Pseudogenes are non- functional genes that accumulated mutations during evolution ► Processed pseudogenes are intron –less, polyadenylated copies of mutated, reverse – transcribed mRNA of cellular genes ► They are assumed to have been created by retrotransposition from cellular RNAs ► Many non-functional copies of tRNA and snRNA genes are flanked by short direct repeats and were likely formed by retrotransposition

. The proposed pattern of expansion of the abundant Alu and B1 sequences found in the human and mouse genomes, respectively ► Both of these transposable DNA sequences are thought to have evolved from the essential 7SL RNA gene which encodes the SRP RNA. On the basis of the species distribution and sequence similarity of these highly repeated elements, the major expansion in copy numbers seems to have occurred independently in mice and humans. geneSRPgeneSRP

Evolutionary considerations ► Mobile elements likely caused many mutations, changing numerous functions during evolution ► Through homologous recombination, they could facilitate gene duplication ► Unequal homologous crossing over formed the globin gene cluster ► Recombination between introns could duplicate domains in proteins ► Exon shuffling could move domains between genes ► Insertion near promoters could affect levels of expression

A comparison of the β-globin gene cluster in the human and mouse genomes, showing the location of transposable elements A comparison of the β-globin gene cluster in the human and mouse genomes, showing the location of transposable elements This human genome contains five β-globin-like genes (orange); the mouse genome has only four. The human Alu sequences (green), and the human L1 sequences (red). The mouse genome contains different but related transposable elements: B1 elements, related to the human Alu sequences (blue triangles), and the mouse L1 elements (related to the human L1, yellow triangles). Because the DNA sequences and positions of the transposable elements found in the mouse and human β-globin gene clusters are so different, it is believed that they accumulated in each of these genomes independently, relatively recently in evolutionary time.genomegenesgenome transposable elementstransposable elementsgenegenomes Alu L1 B1

► ribo.m1v ribo.m1v