Genetics Chapter 15

Insertion into a new location

In one of the first steps of insertion, the transposase makes a staggered cut(5bp) in the target site DNA Transposable element then inserts between staggered ends Host DNA repair machinery fills in the gap opposite each strand overhang (how ?)

piRNAs in animals

In the germ lines of several animal species including Drosophila and mammals, active transposons are repressed through the action of piRNAs (Piwi-interacting RNAs). Like siRNAs, piRNAs are short single-stranded RNAs (26-30 nt in mammals) that interact with a protein complex Once associated, piRNAs guide piwi-Argonaute (a protein complex) to degrade complementary mRNAs does not originate from double stranded mRNA pathway, located in loci called pi-clusters that serve as traps to catch active transposons

Miniature Inverted Repeat Transposable Elements (MITEs)

are nonautonomous DNA transposons Can be formed by deletion of the transposase gene from an autonomous element Can attain high copy numbers (in some grasses)

a large percentage of the dysgenically induced mutations

are unstable; they revert to wild type or to other mutant alleles at very high frequencies.

hich transposable element is used to introduce foreign DNA into the fruit fly Drosophila melanogaster? a. Ac element b. P element c. Alu element d. composite transposons

b. P element

Model organism Maize

produce variably colored kernels Because each kernel is an embryo produced from an individual fertilization, hundreds of offspring can be scored on a single ear, making maize an ideal organism for genetic analysis.

DNA transposons have been modified and used by scientists in two important ways:

(1)to make mutants that can be identified molecularly by having a transposon tag (2) as vectors that can introduce foreign genes into the chromosome.

Tc 1

(DNA transposon) 32 in the worm gene, transposes in somatic but not germ-line cells

Briefly describe the experiment that demonstrates that the transposition of the Ty1 element in yeast takes place through an RNA intermediate.

Boeke, Fink, and their co-workers demonstrated that transposition of the Ty element in yeast involved an RNA intermediate. They constructed a plasmid using a Ty element that had a promoter that could be activated by galactose, and an intron inserted into its coding region. First, the frequency of transposition was greatly increased by the addition of galactose, indicating that an increase in transcription (and production of RNA) was correlated to rates of transposition. More importantly, after transposition they found that the newly transposed Ty DNA lacked the intron sequence. Because intron splicing occurs only during RNA processing, there must have been an RNA intermediate in the transposition event.

Short sequences called IS elements

Can move themselves to new positions -Do not carry genes other than those needed for their environment

transposable elements

Can move to new positions within the same chromosome or even to a different chromosome 50% of our chromosomes

SCID: Severe Combined Immunodeficiency Disease

Caused by a mutation in the gene encoding the blood enzyme adenosine deaminase (ADA) As a result of the loss of this enzyme, precursor cells that give rise to one of the cell types of the immune system are missing Unable to fight infection Treatment: Stem cells can be removed from bone marrow and the wild type copy of the ADA gene is introduced using a retrovirus vector. Transformed cells can then be infused back into the patients. Caveat: Insertion of the viral vector can inactivate important genes and some of the treated patients have developed leukemia

SINE (Short Interspersed Elements)

Do not encode their own reverse transcriptase Nonautonomous, e.g. Alu

Ty elements in yeast are similar to retroviruses because A) both Ty elements and retroviruses utilize an RNA transposition intermediate. B) both Ty elements and retroviruses utilize reverse transcriptase. C) introns are usually found in retroviruses and Ty elements. D) all of the above E) both a and b

E) both a and b

Class 2 DNA transposons

Encode a transposase that cuts the transposon from the chromosome and catalyze the reinsertion at other chromosomal locations P elements (first DNA transposons to be molecularly characterized).

transposons

Not only carry the genes they need for their environment but also carry other genes detected as mobile genetic elements that confer drug resistance (R plasmids ?) consist of recognizable IS elements flanking a gene that encodes drug resistance(not always)

Explain how the properties of P elements in Drosophila make gene-transfer experiments possible in this organism.

P elements are transposable elements found in Drosophila. Under certain conditions, they are highly mobile and can be used to generate new mutations by random insertion and gene knockout. As such, they are a valuable tool to tag and then clone any number of genes. P elements can also be manipulated and used to insert almost any DNA (or gene) into the Drosophila genome. P-element- mediated gene transfer requires inserting the DNA of interest between the inverted repeats necessary for P-element transposition. This recombinant DNA, along with helper intact P-element DNA (to supply the transposase), are then co-injected into very early embryos. The progeny of these embryos are then screened for those that contain the randomly inserted DNA of interest.

R1 and R2 elements of arthropods insert into safe havens

R1 and R2 are LINEs: Insert only into genes that produce ribosomal RNA (rRNA)

Mechanism of transposition in prokaryotes

Replicative method Conservative method

What are safe havens? Are there any places in the much more compact bacterial genomes that might be a safe haven for insertion elements?

Some transposable elements have evolved strategies to insert into safe havens—regions of the genome where they will do minimal harm. Safe havens include duplicate genes (such as tRNA or rRNA genes) and other transposable elements. Safe havens in bacterial genomes might be very specific sequences between genes or the repeated rRNA genes.

safe havens

Successful transposable elements insert into so-called safe havens in the genome For the grasses, safe haven: insert into other retrotransposons Heterochromatin or centromeres (few genes but lots of repetitive DNA)

conservative method

The DNA of the element is not replicated and the element is lost from the site of the original chromosome Also called "cut and paste" mechanism Reaction is initiated by the element encoded transposase Tn 10

"dissociation" (Ds).

The first transposable element Barbara McClintock discovered was a site of chromosome breakage are regulated by an autonomous element called "activator" (Ac) typically located at site of breakage

c-value paradox

The lack of correlation between genome size and biological complexity of an organism

How do plants and animals survive and thrive with so many insertions in genes and so much mobile DNA in the genome ?

Transposable elements can insert into both introns and exons (subject to negative selection) Only the insertion into introns will remain in the population (less likely to cause deleterious mutation) Vast majority can no longer move (inactive) A few elements remain active, and their movement into genes can cause disease e.g; Three separate insertions of LINEs have disrupted the factor VIII gene, causing hemophilia A. At least 11 Alu insertions into human genes have been shown to cause several diseases, including hemophilia B (in the factor IX gene), neurofibromatosis (in the NF1 gene), and breast cancer (in the BRCA2 gene).

Class 1 retrotransposons:

Transpose through RNA intermediates Encode a reverse transcriptase that produces double-stranded DNA copy (from an RNA intermediate)that is capable of integrating at a new position in the genome.

Ty3 is targeted to specific safe havens

Ty3 inserts exclusively near but not in tRNA genes ( do not interfere with the production of tRNAs) Ty3 proteins recognize and bind to subunits of the RNA polymerase complex that have assembled at RNA promoters

Replicative Method

a new copy of the transposable element is generated in the transposition event One copy appears at the new site and one copy remains at the old site Tn 3

Why can't retrotransposons move from one cell to another like retroviruses? a. They do not encode the env protein. b. They are nonautonomous elements. c. They require reverse transcriptase. d. Answer choices a and b are true.

a. They do not encode the env protein. Retroviruses encode the env protein that surrounds the virus. This protein allows the virus to leave the host cell and infect another cell. Without the env protein, the virus is unable to leave the cell. Retrotranspososns do not encode the env protein and hence are unable to move from one cell to another.

Unlike retrotransposons, DNA transposons a. have terminal inverted repeats. b. generate a target site duplication upon insertion. c. transpose via an RNA intermediate. d. are not found in prokaryotes.

a. have terminal inverted repeats. DNA transposons are found in both eukaryotes and prokaryotes and have terminal inverted repeats.

Excision of Ds from the C gene through the action of Ac in cells and their mitotic descendents

allows color to be expressed again, giving a spotted phenotype

c-m(Ds) is unstable when Ac is present in the genome where as

c-m(Ac) always unstable

Approximately what percent of the human genome is derived from transposable elements? a. 10 percent b. 25 percent c. 50 percent d. 75 percent

c. 50 percent. Human genome sequence revealed that around 50 percent of the human genome is derived from transposable elements.

Why are transposable elements found much more often in introns than in exons? a. Transposable elements prefer to insert into introns. b. Transposable elements prefer to insert into exons. c. Transposable elements insert into both exons and introns but selection removes exon insertions. d. None of the above is true.

c. Transposable elements insert into both exons and introns but selection removes exon insertions. Transposable elements can get inserted into both exons and introns; however, the insertions into exons can cause deleterious effects to the host organism and hence are subject to negative selection. Insertions into introns would not cause deleterious mutations and would remain in the population. Introns are an example of a safe heaven, meaning that insertion of transposable elements into these regions would not cause deleterious damage to the host organism.

The major difference between retrotransposons and retroviruses is that a. retrotransposons encode reverse transcriptase. b. retroviruses move from one site in the genome to another. c. retroviruses encode the env gene, which allows them to move from one cell to another. d. None of the above is correct

c. retroviruses encode the env gene, which allows them to move from one cell to another. Retroviruses and retrotransposons both encode gag and pol genes. The pol gene encodes the reverse transcriptase responsible for converting RNA into DNA. However, the env gene is only found in retroviruses, and env protein encoded by this gene allows viruses to move from one cell to another.

Ac

can promote its own transcription found in different location in different plants and animals

non autonomous elements

can transpose only with the help of an autonomous element elsewhere in the genome.

Insertion of Ds in the C gene

creates colorless corn cells

Transposase protein can a. bind to DNA. b. catalyze the excision of a transposable element from a donor site. c. catalyze the insertion of a transposable element into a target site. d. All of the above are correct.

d. All of the above are correct. Transposase protein is encoded by the transposase gene in DNA transposons. It is responsible for the mobilization of the transposase gene from DNA. During transposition, first it binds to DNA and catalyzes the excision of the transposase gene from the chromosome, then it catalyzes its reinsertion at other chromosomal locations/target site.

Why do plants and animals thrive with so many transposable elements in their genomes? a. Most of the transposable elements are inactive due to mutation. b. Active transposable elements are silenced by the host. c. Most transposable elements are inserted in safe havens. d. All of the above are true.

d. All of the above are true. This is mainly due to three reasons. Most transposable elements have accumulated mutations over time and are inactive. As a result, they prevent the production of functional transposase and reverse transcriptase and are unable to move any longer. Active transposable elements are silenced by the host regulatory mechanisms such as RNAi pathway in plants and C. elegans and piRNA pathway in animals. Finally, most transposable elements are inserted in safe havens, such as introns, and do not have a deleterious effect on the host.

Which of the following is true of reverse transcriptase? a. It is required for the movement of DNA transposons. b. It catalyzes the synthesis of DNA from RNA. c. It is required for the transposition of retrotransposons. d. Answer choices b and c are correct.

d. Answer choices b and c are correct. Reverse transcriptase is encoded by the pol gene, which is found in both retroviruses and retrotransposons. It catalyzes the synthesis of DNA from RNA and is required to convert the viral RNA genome into DNA and also for the transposition of retrotransposons.

Which of the following are safe havens for transposable element insertions? a. introns b. exons c. other transposable elements d. Answer choices a and c are correct.

d. Both answer choices a and c are correct. In order to prevent harm to their host, evolutionarily successful transposable elements have inserted into safe havens such as introns, telomeric regions, and other transposable elements.

What is the major reason why the maize genome is much larger than the rice genome? a. Maize has more genes than rice. b. Rice has more genes than maize. c. Maize has more DNA transposons than rice. d. Maize has more retrotransposons than rice.

d. Maize has more retrotransposons than rice. Genome size of organisms usually corresponds to the number of transposable element sequences rather than to gene content. Rice and maize are evolutionary relatives with a common ancestor. The genome size of maize is much larger simply because, over the years, maize has accumulated more retrotransposons.

P elements

discovered as a result of studying hybrid dysgenesis—a phenomenon that occurs when females from laboratory strains of Drosophila melanogaster are mated with males derived from natural populations. In such crosses, the laboratory stocks are said to possess an M cytotype (cell type), and the natural stocks are said to possess a P cytotype.

transposase

enzyme required for the movement of IS elements from one site in the chromosome to another

Bacterial IS

found in E.coli in the gal operon Encode a protein called a transposase begin and end with a short inverted repeat sequences that are required for their mobility

autonomous elements

maize can inactivate a gene in which they reside, cause chromosome breaks, and transpose to new locations within the genome perform these functions unaided

LINE (Long Interspersed Elements)

move like a retrotransposon with the help of an element encoded reverse transcriptase Autonomous, e.g. L1 elements

What is responsible for the additional DNA in the larger genomes ?

repetitive DNA Genome size is usually corresponds to the amount of transposable-element sequences rather than to gene content