Download Supplementary Figure Legends - Word file

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Non-coding RNA wikipedia , lookup

RNA silencing wikipedia , lookup

Short interspersed nuclear elements (SINEs) wikipedia , lookup

Genomic library wikipedia , lookup

Neuronal ceroid lipofuscinosis wikipedia , lookup

Y chromosome wikipedia , lookup

Genomic imprinting wikipedia , lookup

Genetic engineering wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Epigenetics in learning and memory wikipedia , lookup

Neocentromere wikipedia , lookup

Gene expression profiling wikipedia , lookup

Point mutation wikipedia , lookup

Transcription factor wikipedia , lookup

Gene therapy wikipedia , lookup

Epigenetics of depression wikipedia , lookup

Long non-coding RNA wikipedia , lookup

Skewed X-inactivation wikipedia , lookup

Gene nomenclature wikipedia , lookup

Epigenetics of diabetes Type 2 wikipedia , lookup

Genome (book) wikipedia , lookup

Helitron (biology) wikipedia , lookup

Gene wikipedia , lookup

Gene desert wikipedia , lookup

Saethre–Chotzen syndrome wikipedia , lookup

Primary transcript wikipedia , lookup

Epigenetics of human development wikipedia , lookup

Gene expression programming wikipedia , lookup

History of genetic engineering wikipedia , lookup

Gene therapy of the human retina wikipedia , lookup

Microevolution wikipedia , lookup

RNA-Seq wikipedia , lookup

Designer baby wikipedia , lookup

Copy-number variation wikipedia , lookup

Nutriepigenomics wikipedia , lookup

Plasmid wikipedia , lookup

X-inactivation wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

NEDD9 wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Therapeutic gene modulation wikipedia , lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup

Transcript
Supplementary Figure 1 Model for mono-allelic var gene transcription. a, Episomal var
promoter activation. A simplistic display of a P.falciparum nucleus divided into the
transcriptionally incompetent nuclear periphery (red) and a transcriptionally active central
region (green) that extends to the nuclear membrane at one site. This previously described
active perinuclear zone1 includes a telomeric cluster and the proposed exclusive var gene
transcription site (purple). Three theoretical positions of the episomes in 3D7/upsC are shown
(1-3). Postion 1 (transcriptionally inactive zone) does not occur since parasites would be
unable to survive blasticidin-S treatment. The silenced episome assumes position 2
(transcriptionally active region), which allows the hsp86 promoter to transcribe the bsd
resistance gene (black). However, the upsC promoter driving expression of hdhfr remains
silenced due to the interaction of silencing factors with cis-acting var promoter elements.
Activation of the episomal upsC promoter (orange) is only possible if the episome occupies
an exclusive var transcription site (position 3) that is associated with the active chromosomeend cluster at the nuclear periphery. b, Chromosomal var promoter activation. The internal
upsC var gene cluster (blue) on chromosome 12 where integration of pHBupsC occurred
(PFL1960w) is located within the active perinuclear zone due to expression of the plasmidencoded bsd resistance gene (black). In the absence of WR selection var gene transcription
occurs normally and a var gene present in the active chromosome end cluster (blue, white or
yellow) will be transcribed in the exclusive var transcription site (purple) (1, 2). After WRselection, however, the integrated upsC-hdhfr cassette (orange) is activated and displaces the
actively transcribed var gene from the exclusive var transcription site (3).
Supplementary Figure 2 Plasmid copy number and episomal maintenance in WR-untreated
and WR-treated transfectants. a, Plasmid copy number in upsC transfectants. gDNA was
digested with EcoRV and PvuII which allows to discriminate between episomal and
integrated forms. Southern blots were probed with fragments detecting the single copy pfsir2
1
gene (control) and the episomal hdhfr gene. The positions of restriction sites are shown above
each plasmid map. The lengths of the detected fragments are indicated to the right of the
Southern blots. The average plasmid copy number per parasite was determined by
densitometry comparing the signal strength of the hdhfr-containing fragment with that of the
single copy gene pfsir2. b, Plasmid copy number in cam control transfectants. Southern
analysis was performed as above. Note that the growth delay of one generation until full
resistance to WR was observed in 3D7/cam, 3D7/camR and 3D7/camRI (Fig. 1b) reflected the
selection of parasites carrying high plasmid copy numbers (10 to 14) due to the low activity of
the episomal cam promoter. hsp86 5’, heat-shock protein 86 promoter; Pb T, P. berghei
dihydrofolate reductase-thymidilate synthase terminator; upsC, 2.5 kb of upsC upstream
sequence; Pf T, P. falciparum hrp2 3’ terminator; rep20, 0.5 kb rep20 repeat sequence; intron,
0.6kb var intron sequence. Scale bar, 1 kbp.
Supplementary Figure 3 Abundance of hdhfr and bsd transcripts in the control transfectants
3D7/cam, 3D7/camR and 3D7/camRI. The values represent the relative transcript production
per promoter in parasites harvested at 24 to 36 hpi. Values are normalised against differences
in RNA loading (cam signal) and the average plasmid copy number per parasite as determined
by Southern analysis (see Supplementary Fig. 1).
Supplementary Figure 4 Structure of the episomal and integrated plasmid pHBupsC in
3D7/upsC parasites. a, Plasmid map of pHBupsC, and genomic maps of the chromosomecentral var locus PFL1960w, and the integrated pHBupsC plasmid. EcoRV and PvuII
restriction sites used in Southern analysis are indicated. Bold black lines represent the probes
used for hybridisation. The lengths of the restriction fragments detected are shown above each
map. b, Integrated and episomal pHBupsC plasmids and the episomal control plasmid
2
pHBcam analysed by PFG electrophoresis. Blots were probed with pGEM. c, Southern
analysis of genomic DNA confirms integrated and episomal forms of pHBupsC in 3D7/upsC.
S, silenced plasmid-borne upsC promoter; A, activated plasmid-borne upsC promoter.
Supplementary Figure 5 Transgene localisation and knock-down of var transcription in
3D7/upsCR and 3D7/upsCRI. a, Southern analysis of chromosome blots. Parasite
chromosomes were separated by one-dimensional PFGE, blotted and probed with pGEM. In
3D7/upsCR parasites carrying the silenced upsC promoter the plasmid had integrated into
chromosome 12, and was maintained episomally in parasites with the activated upsC
promoter. In 3D7/upsCRI the silenced plasmid had integrated into chromosome 3 and in
parasites carrying the activated form integration occurred into chromosome 12. We confirmed
by genomic Southern analysis that integration into chromosome 12 had occurred by
homologous recombination into the upsC var gene promoter (PFL1960w), and into the rep20
repeat array on the left arm of chromosome 3 (data not shown). S, silenced plasmid-borne
upsC promoter; A, activated plasmid-borne upsC promoter; E, episome. b, var gene
transcription in 3D7/upsCR and 3D7/upsCRI. Total RNA isolated from ring stage parasites was
probed with var exon 2 (see Methods). S, silenced plasmid-borne upsC promoter; A, activated
plasmid-borne upsC promoter.
Supplementary Figure 6 Binding of serum IgG to iRBCs analysed by flow cytometry. IgG
binding was higher to WR-unselected than to WR-selected 3D7/upsC parasites for each of 20
PNG samples tested (fold difference 1.5-8.2). Each sample was tested in duplicate.
1. Duraisingh, M. T. et al. Heterochromatin silencing and locus repositioning linked to
regulation of virulence genes in Plasmodium falciparum. Cell 121, 13-24 (2005).
3