Germline Expression of PElement

1
Germline Expression of P-Element Active
Transposase
2
Figure 23-4. Germ-line-specific expression of
Drosophila P elements an example of
tissue-specific regulation by RNA splicing. (a)
The somatic and germ-line mRNA structures for a
wild-type P element are shown. The germ-line mRNA
is formed by splicing of four exons (numbered 0
to 3) and encodes P transposase. The somatic mRNA
lacks the splice between exons 2 and 3 that is,
the intron between these two exons is retained in
the mRNA. Because the intron between exons 2 and
3 contains a termination codon, the resulting
shorter somatic protein is defective, lacking the
transposase activity. (b) A modified P element
transgene, P ? 2, 3, that lacks the intron
between exons 2 and 3 is depicted. In all
tissues, P? 2, 3 produces an mRNA
indistinguishable from that of the wild-type P
element, resulting in P transposase activity in
both germ line and soma.
3
Figure 8-37. Generation of transgenic fruit flies
by P-element transformation. The P element, a
mobile genetic element, can move from one place
in the genome to another. This movement
(transposition) is catalyzed by transposase,
which is encoded by the P element the 3' and 5'
ends of the P element are recognized by
transposase and are required for transposition to
occur. To produce transgenic fruit flies by this
method, the functionally different regions of the
P element are incorporated into two different
bacterial plasmids. The donor plasmid contains
three necessary elements the transgene (orange)
a marker gene (green) used to indicate flies in
which the plasmid DNA is transposed to a
recipient chromosome and both ends of the P
element (dark purple) 3' P and 5' P flanking
the other two genes. It does not contain
transposase. In this example, the marker is the
dominant w allele, which confers red eye color.
The red bracket indicates the segment of the
donor plasmid that can transpose into the fly
genome. The other plasmid carries the P element
(encoding transposase) with mutations in one end,
which prevent it from transposing. The two
plasmids are co-injected into blastoderm embryos
homozygous for the recessive w- allele, which
confers white eye color. Transposase synthesized
from the gene on the P-element plasmid catalyzes
transposition of the donor plasmid DNA into the
fly genome. Because transposition occurs only in
germ-line cells (not in somatic cells), all the
G0 adults that develop from injected embryos have
white eyes. Mating of these flies with white-eyed
flies will yield some G1 red-eyed progeny
carrying the transgene and the marker allele (w)
in all cells.
4
The White Locus

• First described by Morgan, 1910 and is most
  intensively studied (2348 references).
• X-linked important for sex linkage studies.
• Many alleles (1352) and allele phenotypes (10)
  ranging from white (null mutants) to red/ brown.
• Non-essential, ATP-binding cassette (ABC)
  transporter.
• Cell autonomous, therefore utilised extensively
  as a marker in mosaic flies.
• Has revealed several important genetic phenomena
• Position effect variegation (PEV).
• Dosage compensation.
• Sex linkage
• Transvection effects
• Genetic instability due to transposable element
  insertions

5
Position Effect Variegation (PEV)
Now accepted that heritable changes in gene
expression can occur without accompanying changes
in DNA sequence.

• PEV first described by H.J. Muller in 1930.
• Undertook X-ray mutagenesis and found a new class
  of mutations that could simultaneously affect the
  expression of several genes in the same genomic
  location.

• Mutations of the white eye colour gene showed
  striking cell-to-cell variation (mosaic
  phenotype).
• Notch, linked to white also displayed variation.
• The mosaic phenotype was caused by a chromosomal
  rearrangement/ position effect displacing white
  from its normal position.

6
Position Effect Variegation (PEV)

• Schultz a few years later examined polytene
  chromosomes of 13 different variegating mutants.
• In all cases an inversion or translocation
  changed the position of the gene from a
  euchromatic to heterochromatic position.
• Some rearrangements gave large patches of red
  facets adjacent to large patches of white.
• Conclusion Decision on expression of white made
  early during tissue development and maintained
  through multiple cell divisions.
• Gene is not mutated movement of the rearranged
  allele away from heterochromatin can restore
  expression.

7
Euchromatin and Heterochromatin

• Heterochromatin
• Deeply stains with cytochromic markers.
• Condensed throughout the cell cycle.
• Contains diverse repetitive DNA and unique
  sequences (includes rDNA).
• Contains some specific genes that are only
  expressed in this location.
• Euchromatin
• Diffuse and difficult to visualise during many
  stages of the cell cycle.
• Contains housekeeping and single copy genes.

About 30 of female and 35 of male genome is
heterochromatin. (All of Y, 40 of X, 25 of
chromosomes 2 and 3 and most of 4.
8
PEV Model

• In 1939, Schultz offered the first model to
  explain the results.
• Observations showed that whenever a gene more
  distant from a breakpoint showed a mutant
  phenotype (white), the gene closer did also
  (Notch).
• An inactivation process spreads from the
  heterochromatic breakpoint along the chromosome.
• Visually the area became darkened, disarranged
  and sometimes completely heterochromatic.
• The oozing model was proposed in the late
  1980s depends on heterochromatic-specific
  proteins propagating continuously along the
  chromosome and blocking access to transcription
  factors.

9
Figure 9-51. Position effects on gene expression.
Position effects can be observed for the
Drosophila white gene. Wild-type flies with a
normal white gene have red eyes. If the white
gene is inactivated by mutation, the eyes become
white (hence the name of the gene). In flies with
a chromosomal inversion that moves the white gene
near a heterochromatic region, the eyes are
mottled, with red and white patches. The white
patches represent cells where the white gene is
silenced and red patches represent cells that
express the white gene. The difference is thought
to arise from variations in how far along the
chromosome the heterochromatin spreads early in
eye development. Once established, the state of
white expression is heritable, producing patches
of many cells that express white as well as
patches of cells where white is silenced. (After
L.L. Sandell and V.A. Zakian, Trends Cell Biol.
210-14, 1992.)
10
Figure 9-65. Position-effect variegation in
Drosophila. (A) Heterochromatin (red) is normally
prevented from spreading into adjacent regions of
euchromatin (green) by special barrier sequences
of unknown nature. In flies that inherit certain
chromosomal translocations, however, this barrier
is no longer present. (B) During the early
development of such flies, the heterochromatin
now spreads into neighboring chromosomal DNA,
proceeding for different distances in different
cells. The spreading soon stops, but the
established pattern of heterochromatin is
inherited, so that large clones of progeny cells
are produced that have the same neighboring genes
condensed into heterochromatin and thereby
inactivated (hence the "variegated" appearance of
some of these flies see Figure 9-51B). This
phenomenon shares many features with X-chromosome
inactivation in mammals.
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Factors Affecting PEV

• The presence of Y chromosomes.
• Absence of Y enhances variegation, increases in
  Y reduce variegation. Y probably titrates out a
  factor or factors involved in making
  heterochromatin.
• Unlinked genes
• Enhancers of variegation e.g. E(var)7 on
  chromosome 2L ttranscriptional activator of
  heterochromatin proteins.
• Suppressors of variegation e.g. Su(var)7 on
  chromosome 3L heterochromatin protein.

12
Implications of Observations

• Euchromatin can be converted to heterochromatin
  when chromosome structure is disturbed.
• Conversion to heterochromatin leads to gene
  silencing.
• Disruption of chromosome structure causes a
  breakdown in the extent of heterochromatinisation.
  
• Extent of heterochromatin spreading is random or
  stochastic following a chromosome break.
• There is eventual stabilisation of
  heterochromatin spreading as daughter cells show
  stable epigenetic inheritance of the inactivation.

13
Implications of Observations

• There must be cis-acting signals in the DNA
  (start-stop)
• Heterochromatin initiation signals.
• Heterochromatin propagation signals.
• Heterochromatin termination signals.
• Trans acting factors
• Proteins that are unique to heterochromatin and
  euchromatin must exist.
• Chromatin insulators or boundary elements
  composed of both cis-acting and trans-acting
  factors must occur to insulate normal euchromatin.

14
Some Observations Against the Model

• Heterochromatin is accessible to transcription
  factors rDNA and heterochromatin-specific
  sequences are transcribed.
• Cis-spreading of heterchromatin does not explain
  all observations
• Gene inactivation does not always correlate with
  the amount of heterochromatin.
• The frequency of silencing of the more distal
  gene from the heterochromatin can be higher than
  the more proximal gene.
• There is evidence that heterochromatin may act in
  trans to suppress variegating alleles.
• See Wakimoto (1998) Cell 93, 321-324.

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Figure 1. Models to Explain the Effect of
Heterochromatin on Gene Expression A euchromatic
gene placed next to heterochromatin is expressed
(A) or repressed (B and C) depending on the
extent of cis-spreading of heterochromatin
proteins. In the conventional view of PEV (B),
the invading heterochromatin proteins (H-Raps)
impose a closed chromatin state onto the
euchromatic gene, blocking access of the
transcriptional machinery. Alternatively, the
proteins could interact with transcription
factors and form a repressor complex at
euchromatic gene promoters (C). H-Rap proteins
act differently in the context of heterochromatic
gene promoters. They may facilitate transcription
by interacting with other factors to form an
activating complex (D), or by mediating
long-distance communication between enhancers and
promoters via proteinprotein interactions (E).
16
A Reexamination of Spreading of Position-Effect
Variegation in the white-roughest Region of
Drosophila melanogaster Paul B. Talbert and
Steven HenikoffGenetics, Vol. 154, 259-272,
January 2000
17
Figure 1. Schematic diagrams of the wm4, wmMc,
and w51b inversions. The top line shows a
magnified view of the region around the w gene.
Other solid lines represent the X chromosome,
which is not drawn to scale. Dashed lines
indicate the location of type I and 359-bp
repeats. Open boxes, ribosomal DNA repeats
filled boxes, other heterochromatin vertical
arrows, inversion breakpoints horizontal arrow,
the w transcription unit. Data from APPELS and
HILLIKER 1982 and TARTOF et al. 1984 .
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Figure 3. Variegated eye phenotypes of X/0 males.
Note the rough patches in the eyes of the McT/0,
4LMcR/0, and 51b/0 flies (arrowheads).