Source of D. littoralis fosmid clones

Size: px

Start display at page:

Download "Source of D. littoralis fosmid clones"

Audrey Burns
5 years ago
Views:

1 Source of D. littoralis fosmid clones Libby Slawson Bio 4342 January 28, 2004 Overview What is a fosmid? Library construction Selection of target genes Probing the Library Confirming clones What is a fosmid? A cloning system based on the E. coli F factor. These clones have an average insert size of 40 Kb, with a very small standard deviation. -NCBI glossary 1

Library Construction Fosmid library we are using was created by the BACPAC Resource Center Genomic DNA was partially digested by MboI (four-cutter), size selected, and cloned into the BamHI site of

2 Library Construction Fosmid library we are using was created by the BACPAC Resource Center Genomic DNA was partially digested by MboI (four-cutter), size selected, and cloned into the BamHI site of pfos1 NNGATCNNNNNNNNNNGATCNN NNGATCNNNNNNNNNNCTAGNN MboI NGGATCCN NCCTAGGN MboI BamHI NGGATCNNNNNNNNNNGATCCN NCCTAGNNNNNNNNNNCTAGGN? Packaging into Phage Why fosmids? This library was available on a gridded membrane Filters were spotted with 36,864 colonies, representing 18,432 individual clones, spotted in duplicate Order by number! 2

Our research goal To compare sequence from the dot chromosome of D. melanogaster with D.

pseudoobscura, a species 25-30 my diverged from D. melanogaster Last spring, Rachel did a BLAST comparison using cdna sequences from all the genes from the dot chromosome of D.

pseudoobscura from the Baylor website She looked for >200bp chunks of genes that were very highly conserved (>80%) and designed PCR primers using Primer3 (http://www.broad.mit.

3 Our research goal To compare sequence from the dot chromosome of D. melanogaster with D. littoralis Selection of target genes by Rachel Shevchek Small (dot) chromosome of many Drosophila species is known to have similar genes (Podemski, 2001*) Sequencing has recently been completed for D. pseudoobscura, a species my diverged from D. melanogaster Last spring, Rachel did a BLAST comparison using cdna sequences from all the genes from the dot chromosome of D. melanogaster to the genomic sequence of D. pseudoobscura from the Baylor website She looked for >200bp chunks of genes that were very highly conserved (>80%) and designed PCR primers using Primer3 ( *Podemski, Ferrer, Locke (2001) Whole arm inversions of chromosome 4 in Drosophila species, Chromosoma 110, PCR primers designed for 30 genes total Fourth Chromosome Map: Conserved Genes Between D. melanogaster and D. psuedoobscura 25kb PlexB pan Ank CG31998 Ci CG kb Syt7 BEST CG2052 Hcf 425kb CaMKI CG11533 zfh2 Thd1 CG1970 onecut 625kb Eph CG1732 CG11360 ey bt1 bt2 CG31992 myoglianin 825kb CG11152 CG11148 Glu-Ra CG11093 toy unc kb PlexA ATPsyn CG32018 Arf102F 3

PCR generation of probe template Rachel did PCR using D.

were used as template for the probe reaction Probing

as template in a Klenow reaction with 32 P labeled dctp

Filters were probed and washed at low stringency

After exposing the filters to film, Elmer picked

4 PCR generation of probe template Rachel did PCR using D. melanogaster genomic DNA for template The PCR products were used as template for the probe reaction Probing the libraries by Elmer Kellmann PCR products were used as template in a Klenow reaction with 32 P labeled dctp Three probes were pooled to use in one hybridization Filters were probed and washed at low stringency because of large amount of distance between species After exposing the filters to film, Elmer picked positive clones and ordered from the BACPAC center Results 52 positive clones 4

hybridization, we didn t know which clone

ordered the wrong clone BACPAC center could have

blot Southern blot Overlapping restriction fragments

5 Confirming clones Why confirm? Methods used: Since we pooled several probes in one hybridization, we didn t know which clone corresponded to which probe/gene We could have ordered the wrong clone BACPAC center could have sent us the wrong clone Colony hybridization Slot blot Southern blot Overlapping restriction fragments In situ hybridizations Something could have gotten labeled wrong Colony hybridization Slot blots 5

Southern blots We needed to do preliminary restriction mapping to send on to the GSC

Overlapping restriction maps In situ hybridizations Ten fosmids in process of being

Mary-Lou Pardue at MIT Will not tell us what gene the fosmid contains, but will tell

6 Southern blots We needed to do preliminary restriction mapping to send on to the GSC Transfer gel to filter after taking a picture and blot Ended up not working so well Overlapping restriction maps In situ hybridizations Ten fosmids in process of being probed against polytene chromosomes of D. littoralis Being done in the lab of Dr. Mary-Lou Pardue at MIT Will not tell us what gene the fosmid contains, but will tell us where some of the DNA from the fosmid is localized *example in situ, not our results 6

Confirmed clones 18 of the original 52 fosmid clones were sent on to the GSC Successful libraries were made from 10 clones by Library Core You are working on the subclones picked from these libraries

7 Confirmed clones 18 of the original 52 fosmid clones were sent on to the GSC Successful libraries were made from 10 clones by Library Core You are working on the subclones picked from these libraries The other 8 clones will be finished by the instructors and new techniques will be used to find clones for next year Map of clones to date 25 kb pan 2 GSC Ank CG32000 CG kb BEST Hcf 2 GSC CG GSC 425 kb CaMKI CG11533 Thd1 1-2 GSC CG1970 onecut 1 GSC 625 kb 450 kb CG1732 CG31992 bt2 1 GSC 2 GSC 825 kb Glu-Ra CG11093 toy 1025 kb 925 kb ATPsyn Arf 2 GSC 950 kb 975 kb 1000 kb 1025 kb 1050 kb Questions? 7

8 Your fosmid clones XAAA103 Arf102F, CG11093 XAAA106 Overlapping RE maps XAAA11 ATPsynB XAAA112 Hcf, CG2052 XAAA113 Hcf, CG2052 XAAA121 BEST XAAA122 BEST XAAA72 Onecut XAAA73 CG1970, CG31998 XAAA83 Overlapping RE maps Reminder: Hybridization means sequence similarity, not genetic homology! 8

The fourth chromosome: targeting heterochromatin formation in Drosophila. Drosophila melanogaster chromosomes

The fourth chromosome: targeting heterochromatin formation in Drosophila Drosophila melanogaster chromosomes modified from TS Painter, 1934, J. Hered 25: 465-476. 1 DNA packaging domains HATs Euchromatin