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unc-96 belongs to a major class of muscle affecting
genes that has an “Unc” or uncoordinated phenotype.
To date, there are three alleles for unc-96(su151, r291, and sf18).
This class of genes paralyzes or generally disturbs muscle organization
of fully developed adult worms. unc-96 is required for proper myofibril
organization and has a polarized light phenotype that is characterized
by highly birefringent "needles" near the ends of muscle
cells and abnormal localization of thick and thin filaments. unc-96
had previously been mapped to the left arm of the X chromosome.
Using two factor mapping, three factor mapping, deficiencies, duplications,
and SNP mapping, unc-96 has been shown to lie between unc-1 (specifically,
to the right of cosmid T26C11) and dpy-3, all of which is an interval
less than or equal to 400 kb containing nine overlapping cosmids,
two PCR fragments (to cover gaps), and two YACs. To map unc-96 to
a finer level, cosmids and YACs were used in transgenic rescue attempts
and so far, results show that the region 1,952,044 to 2,144,449
(192 kb) and the region 1,839,283 to 1,875,917 (36 kb) does not
contain the gene in question. We will continue transgenic rescue
with cosmids, restriction fragments and/or PCR fragments until we
obtain rescue with a segment that contains the transcriptional unit
of a single predicted gene.
C. elegans makes a great model for biological study
because of its simplicity, transparency, ease of cultivation in
the laboratory, short life cycle, suitability for genetic analysis,
and small genome size (Wood eds., 1988). Because of these attributes,
the nematode is a favorable organism in which to study muscle. In
particular, the optical transparency of the worm allows easy evaluation
of muscle structures and large numbers of animals can be handled,
which makes screening for rare mutations practical. Due to the conservation
of muscle structure and proteins, the information harnessed from
the study of C. elegans can be extrapolated to other animals, including
man. For example, the analysis of muscle protein function in C.
elegans is leading to advancements in our understanding of genetic
diseases such as muscular dystrophy. The genome sequence of C. elegans
is found to have genes that encode homologs for many of the components
of the mammalian dystrophin glycoprotein complex. This has important
implications since the dystrophin gene is defective in Duchenne’s
and Becker’s muscular dystrophies. Researchers hope to accelerate
the search for a cure of Duchenne muscular dystrophy with the knowledge
gained from the human genome project and C. elegans genetics.In
addition, genetic analysis in the nematode has provided the larger
community of muscle researchers with the first cloned genes and
functional data on many fundamental components of muscle, i.e. the
complete sequence of a myosin heavy-chain gene (Wood ed., 1988).
Muscle research in the worm has also lead to the development
of useful molecular genetic strategies and methods. In the nematode,
the muscles used for locomotion are obliquely striated and are located
in the body wall. The fundamental repeating unit that is responsible
for muscle contraction is the sarcomere. The sarcomere is composed
of a bundle of myosin-containing thick filaments interwoven with
a bundle of actin-containing thin filaments. Throughout the muscle
cell, the thin filament attachment structures, called the dense
bodies (analogous to the Z-discs in vertebrate muscle) and the thick
filament cross-linking structures, the M-lines, are anchored to
the muscle cell membrane. This permits the force of contraction
to be transmitted through the cell membrane, the basement membrane
and the hypodermis, to the overlying cuticle and results in movement
of the whole animal. Much is known about the components of the sarcomere,
but relatively little is known about how myofibrils are assembled,
or how myofibrils are maintained during the stress of repeated muscle
activity.
Numerous genes control the organization of sarcomeres
and their function. In particular, there is a major class of muscle
affecting genes in C. elegans that has an “Unc” or uncoordinated
phenotype. These muscle Unc genes paralyze, retard the movement,
or generally disturb muscle organization of fully developed adult
worms.
Our research focuses on mapping the unc-96 gene, which
is required for proper myofibril organization. unc-96(su151) has
a polarized light phenotype that is characterized by highly birefringent
"needles" near the ends of muscle cells, without any definite
A or I bands. In addition, there are extra collections of thin filaments
or intermediate filaments, and masses of thick and thin filaments
in abnormal locations. Our goal is to map unc-96 to a finer level.
When this is achieved, we will then move onto determining the nature
of the protein encoded by unc-96 and its intracellular location
and interacting partners. Thus far, unc-96(su151) had previously
been roughly mapped to the left arm of the X chromosome. By two
factor mapping, unc-96 had been placed to the left of dyp-3.
We performed three factor mapping to show that unc-96
lies between unc-1 and dyp-3 (approximately three map units apart).
We then used deficiencies and duplications to narrow unc-96 to approximately
one map unit (T. Tinley, K. Mercer, G. Benian, unpub. data). We
found the left most physical limit and the right most physical limit
for the region containing unc-96 by finding the right breakpoint
of meDf2 and the right breakpoint for meDf6, respectively. Using
a positive and negative control, PCR was performed to amplify a
small fragment within cosmids lying in the region between unc-1
and lin-32 (both are cloned genes; dpy-3 has not yet been cloned).
The presence of a product meant that the strain was not deficient
in the given position and therefore ends near that position on the
physical map. The breakpoints of meDf2 and meDf6 narrowed the unc-96
region to 1Mb.
In the current study, we aim to continue mapping unc-96
to a finer level using “single nucleotide polymorphism”
(SNP) mapping (Hill et al, 2000). To achieve this, we propose the
following objectives:
- map unc-96 to the level of several (2-10) overlapping cosmids
with eight SNPs
- use cosmids in transgenic rescue experiments; if successful,
perform more rescue attempts with ever smaller segments
- use restriction fragments and/or PCR fragments until we obtain
rescue with a segment that contains the transcriptional unit of
a single predicted gene
- use overlapping YAC clones or PCR generated fragments to map
the four sub-regions that have no cosmid coverag.
Nematode Strains mnDp66(X;1) unc-1(e538) unc-96(r291)
dyp-3(e27) CB4856 (Hawaiian strain) Construction of Triple Mutant
A triple mutant was needed in order to isolate recombinants by using
the loss of an easily indentified, morphological marker. For this
study, we chose unc-1 and dpy-3 which flank unc-96. The triple was
made by crossing mnDp66 unc-1 males to unc-96 dpy-3 hermaphrodites.
F3 UNC-1 DPY-3 recombinants were isolated and viewed by polarized
light to confirm the present of UNC-96. Acquisition of Recombinants
Hawaiian males were mated to the triple, unc-1 unc-96 dpy-3 hermaphrodites.
In the F2, unc-non-dpys or dpy-non-uncs recombinants were isolated
and the recombinant chromosome homozygosed by allowing self-fertilization.
Once the homozygous recombinants were identified, we allowed the
worm to reproduce several generations and then isolated the genomic
DNA. Making Genome DNA for Sequencing Genomic DNA will be produced
using a standard C. elegans procedure, which involves phenol/chloroform
extraction and ethanol precipitation. Cosmid Prep for Injection
Cosmids will be ordered from the Sanger Center, Cambridge, England.
Cosmid DNA will then be isolated using a commercially available
DNA isolation kit (Qiagen, Inc.). This DNA will then be diluted
and used for injection, together with the transformation marker
rol-6. Lines will be recovered from F1 rollers, their muscle assessed
for the Unc-96 phenotype by polarized light microscopy.
All alleles of unc-96 have the characteristic appearance
of “needles” near the ends of the muscle cell under
polarized light. Both unc-96(r291) and unc-96(su151) have “needles”
whereas N2 (wild type) does not.
Cross sections of body wall muscle from N2 (wild type)
and unc-96 mutant alleles, su151, sf18, and r291 were examined by
electron microscopy. Functionally analogous to Z-lines in vertebrate
muscle, dense bodies are finger-shaped structures that project from
the plasma membrane into the cytoplasm. One primary role of dense
bodies is believed to be attachment and alignment of the thin filaments.
M-lines maintain the alignment of thick filaments. Thick and thin
filaments are seen as large and small dots respectively. We observed
disrupted muscle structure in the mutant alleles, all of which had
irregular interspersion and abnormal localization of thick and thin
filaments. In addition, dense bodies are shortened and often broken.
By using deletions and duplications, we were able
to show that unc-96 lies within an approximately 1.5 map unit region
on the left arm of the X chromosome. This region lies to the right
of the right breakpoint of deficiency meDf2 and to the left of the
left edge of the duplication yDp14.
Breakpoints of the deficiencies meDf2 and meDf6 (~1
Mb): the right breakpoint for meDf2 lies within cosmid D1005, and
the left breakpoint for meDf6 lies within cosmid T14F9. SNP mapping
has limited the region to progressively smaller regions. First,
we learned that the unc-96 region lies to the right of cosmid K06A9
(approx. 800 kb region), and then later, we learned that unc-96
lies to the right of cosmid T26C11 (approx. 400 kb region).
YACs and PCR fragments (to cover gaps) in the unc-96
region were injected into unc-96 mutant worms with rol-6 as a marker
to obtain lines. The lines were then screened under polarized light
to see if rescue had occurred (restoration to wild type polarized
light appearance). No rescue out of a minimum of four lines indicates
that unc-96 does not likely lie within these segments. The X indicates
the regions where rescue was not obtained.
We will continue to microinject with the remaining cosmids, YACs,
and PCR fragment in the remaining region. When rescue is successful,
we will sequence the area and conducts expirements that will show
us for sure if the area suspected does contain our gene. We will
also investigate how the gene interacts with other genes and analyze
the gene products.
I would like to thank the Benian Lab for their support and involvement.
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No.52003071.
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