Our laboratory at Smith College (Northhamptom, Massachussetts) has constructed
a new Onchocerca volvulus cDNA library that has been constructed
using 5,000 L3 larvae collected by Sara Lustigman in the Republic of Cameroon
and maintained by Milan Trpis in the EMCF L3 bank at Johns Hopkins. The
methods used to construct this cDNA library were developed first using the
lymphatic filarial parasite Brugia malayi as a model system, since
L3 larvae from Brugia are easier to obtain and less expensive.
The method used was a traditional cDNA library construction (i.e. not using
PCR) designed to optimize the amount of mRNA isolated from only 5,000 larvae.
Larvae frozen in liquid nitrogen were pulverized into powder and then resuspended
in guanidinium solution. polyA+ mRNA was isolated and used as the template
for cDNA synthesis and cloning into the lambda vector UniZap XR (Stratagene).
In this unidirectional vector, the EcoRI end of the inserted cDNA always
represents the upstream (5') end of the original mRNA.
The O. volvulus L3 cDNA library was shown to have an unamplified
titer of 3.6 x 10<sup>5 </sup>and an amplified titer of greater
than 1010. Only 4% of the clones in the library are rRNA sequences. Based
on the analysis of over 300 randomly selected clones, the average insert
size is estimated at 1,000 base pairs. All 300 of these clones have been
partially sequenced at their 5' end to initiate an O. volvulus EST
(expressed sequence tag) database. All of the ESTs have been compared to
nucleic acid and protein sequence databases using Blastn and Blastx searches
available through NCBI (National Center for Biotechnology Information).
65% of the ESTs show a significant 'hit' to a gene or protein sequence
already present in the database. Of these 'hits', 80% are to genes of known
function, while 20% are to genes of unknown function. 35% of the ESTs show
no significant 'hits' to any sequences present in any of the databases.
Several of the ESTs have been identified as copies of genes already cloned
from O. volvulus by other investigators. All of the EST data has
been deposited in the dbEST database curated at the NCBI.
Over 100 new genes of interest have already been identified using this
EST approach. Some of these genes include calmodulin, myosin heavy chain,
troponin, various heat shock proteins, a homolog of macrophage migration
inhibitory factor, and many others. In addition, in experiments done in
collaboration with Dr. James McCarthy and Dr. Thomas Nutman at the NIH,
about 50 clones have been identified by screening with sera from infected
or immune patients. Some of these genes have been cloned previously by
other investigators (Ov-17, Ov-33, onchocystatin, cyclophilin, cuticular
collagen, intermediate filament), but nearly 50% are either new genes (i.e.
no hits in the databases) or genes never before cloned from nematodes.
Many of these genes code for proteins that should be evaluated for their
vaccine potential.
This L3 library has already been distributed to seven groups including
the Nutman, Weil, Scott, Bianco, Lustigman, Perler, and Unnasch laboratories.
Many of these groups have already identified new genes of interest from
this library. For example, the Bianco laboratory has identified a chitinase
gene from this library with 67% identity to an A. viteae chitinase previously
cloned in their laboratory. Based on all of these data, the O. volvulus
L3 cDNA library was judged to be suitable for distribution to other investigators.
Note from editor: Steve Williams may be contacted at Smith College
by the following:
Dr. Steven Williams phone: (413) 585-3826
Clark Science Center SR 407 FAX: (413) 585-3786
Department of Biological Sciences e-mail swilliams@smith.smith.edu
Smith College
Northhampton, MA 01063