Worm Breeder's Gazette 14(4): 14 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | 4444 Forest Park Blvd., St. Louis MO 63108 |
| 2 | Wellcome Trust Genome Campus, Hinxton, Cambs CB10 1SA, UK. |
We have constructed C. elegans and C. briggsae genomic DNA
libraries in the pFOS1 "fosmid" vector system. Fosmids are low copy
number cosmid-like vectors based on the E. coli F factor replicon (Kim
et al. 1992 NAR 20 1083 - 1085). In our hands fosmid clones offer two
main advantages. First, randomly selected clones appear to undergo
deletion events at a rate much reduced compared to similarly chosen
cosmid clones. Second, the yields of DNA obtained from fosmids, while
reduced compared to some cosmids, appear reproducible between clones.
Both libraries were constructed by ligation of Nde II
partially digested genomic DNA into BamHI digested pFOS1 followed by
packaging into lambda phage and plating on XL1 Blue MR cells.
Approximately 18,000 clones from each library were individually picked
into 384 well plates for preservation in glycerol, and these clones
were then used to construct high density filters for hybridization.
Each filter is 22 cm square and contains all 18,000 clones
(approximately 7 X coverage for the genome) with each clone spotted
twice on the filter to control for false positives and background.
Robotic picking of the clones into 384 well format and construction of
the high density filters were performed courtesy of Genome Systems
Inc. (St. Louis) who are offering both the high density filters and
hybridization based screening of them as services. Interested labs
can contact Genome Systems at genome@mo.net.
The C. elegans fosmid library has been characterized by
fingerprint analysis of clones selected at random and by direct
probing of the high density filters using probe DNAs donated by
C. elegans researchers or generated in our labs. Fingerprint data has
been incorporated into ACeDB. Fosmid clones are designated in the
database by the letter "H". As a result of our analysis, fosmid
clones have been identified which extend into gaps in both
cosmid-dense and cosmid-poor regions. However, our results indicate
that some regions not represented in cosmid clones will not be
represented in fosmid clones. Additionally, stable fosmids replacing
deleted cosmids have been recovered. As we continue to characterize
the library and identify clones for sequencing we are willing to
receive probe DNAs from the community and to fingerprint fosmid clones
identified in other labs.
During characterization of the C. briggsae fosmid library we
identified a number of C. briggsae fosmid clones using C. elegans
probe DNAs provided by, among others, P. Hoppe, C. Hresko and P. Tan.
Probe DNAs were used in hybridization experiments to the high density
filters described above and resulting positive clones were
fingerprinted and clones selected for sequencing. These sequences
will, as with C. elegans sequences, be available on our WWW site
(http:// genome.wustl.edu/gsc/). As part of our larger effort to
construct a physical map of the C. briggsae genome and generate
several megabases of C. briggsae genomic sequence we are willing to
consider requests from the community for assistance in mapping and
sequencing cosmid or fosmid clones. As a cautionary note, a small
number (~15) of the gridded clones appear to be contaminated with
vector DNA; we have observed that the cleanest hybridization results
to the high density filters are achieved with probe DNAs that are
completely free of vector. Questions concerning the C. briggsae
fosmid library should be directed to Marco Marra
(mmarra@watson.wustl.edu). Questions concerning the C. elegans fosmid
library should be directed to Stephanie Chissoe
(schissoe@watson.wustl.edu). For a fosmid DNA isolation protocol see
http://genome.wustl.edu/gsc/manual/protocols/ - BAC / PAC / Fosmid
prep.