Biology Reference
In-Depth Information
As well as providing sequence tags, ESTs can be used to map human
genes, without knowledge of the gene's function. A sequence tagged site
(STS) is created from an EST by mapping the EST using radiation hybrid
panels. Radiation hybrid panels are collections of somatic cell hybrid clones,
usually hamster-human hybrids, in which irradiated human genomic DNA
was fused with hamster cells. Each hamster cell line incorporated different
small fragments of human DNA, so that only human DNA markers that
are physically very close to one another are likely to be present in the same
hamster line. Thus, detection of an EST in a cell line indicates that it is close
to the markers that are known to be present in that cell line.
3.1 Cochlear cDNA Libraries and Cochlear Expressed
Sequence Tags
Cochlear ESTs have several uses. They provide sequence tags that enable
identification, mapping and cloning of cochlear-expressed genes, both those
that are unique to the cochlea, and those expressed in many or all tissues.
This may prove to be a particularly valuable tool because a number of deaf-
ness loci have been mapped by linkage analysis (Mueller, Van Camp, and
Lench, Chapter 4), but the genes have yet to be cloned.
Certainly, human cochlear tissue is a notoriously difficult tissue from
which to obtain good quality mRNA for the generation of cDNA libraries.
The first human cochlear cDNA library was created by Robertson et al.
(1994). This library, known as the Morton Fetal Cochlear cDNA Library, has
resulted in the generation of over 4,000 human cochlear ESTs from 3,200
individual clones (Skvorak et al. 1999). All the cochlear EST sequences can
be found in dbEST, and all the clones are commercially available.
In addition to the Morton human cochlear cDNA library, a number of
rodent and avian cochlear cDNA libraries have also been developed.
Guinea pig or chick cochleae, while small and difficult to isolate, are still
far easier to obtain than human cochleae. Additionally, because cochlear
tissues can be obtained from animals at various time points, developmen-
tal processes can be studied in animal models that cannot be attempted
using human tissues.
3.1.1 Known Genes Expressed in the Human Cochlea
The sequences of all of the ESTs generated from the Morton fetal
cochlear cDNA library were compared with sequence data in the various
GenBank databases. This analysis revealed that 33% were very similar
to previously identified human genes (Skvorak et al. 1999). In all, signifi-
cant similarity to 517 known human genes was found. Because the library
had not been subtracted or normalized in any way, many of the known
genes were “housekeeping” genes. A complete list can be found at
http://hearing.bwh.harvard.edu. Several of the cochlear ESTs were from
