DOI Page / Citation link:
https://doi.org/10.11588/diglit.48120#0029
Abstracts
11
RNA-binding domain of the 70 K U1 snRNP protein. This region encompasses
the octamer sequence which has been observed in a variety of proteins associated
with RNA (Dreyfuss et al. 1988). In addition, Ro protein contains a potential
“zinebinding finger’’ motif proposed to be involved in nucleic acid-or protein-
protein interactions. The involvement of the putative RNA recognition motif
(RRM) and “zinc finger” motif in binding hY RNA is being determined. Muta-
tions in the RRM and “zinc finger” regions as well as mutations throughout Ro
protein have been created. The recombinant proteins, overexpressed in bacteria or
in vitro translated, have been used in reconstitution assays with hY 1 and hY 3
RNA transcripts in order to define the RNA-binding domain of the 60 kD Ro pro-
tein and to determine the stoichiometry of the complex. Gel shift assays using
mutated Ro translation products and hY 1 RNA have shown that the putative
zinc-finger is not required for binding to hY RNA or DNA. However, the octamer
sequence within the RRM, is required for the interaction of 60 kD Ro with hY
RNA. The sites on the hY RNAs required for interaction with the 60 kD Ro prote-
in are being determined using various mutant hY RNA transcripts reconstituted
with full-length Ro protein. Preliminary studies suggest that sequences at the 3'
end of the hY 1 RNA are not involved in binding Ro protein.
Monoclonal Autoantibodies Against Nuclear Proteins
S. Dyos, L. Alldridge, G. Dealtry, and M. O’Farrell
Department of Biology, University of Essex, Wivenhoe Park, Colchester,
Essex CO4 3SQ, UK
We are investigating the role of nuclear proteins in the control of cell prolifera-
tion. In order to study the structure and function of individual proteins and also
to identify interactions between different proteins we have initiated a programme
to generate monoclonal antibodies to macromolecular components of the
nucleus. We are using autoimmune strains of mice as a biological model system
in which to generate monoclonal antinuclear antibodies. Splenocytes from NZ
B/W Fl female mice (between 4 and 5 months of age) were fused with SP2/0
myelomas . Hybridomas secreting antibodies that recognise the nucleus of fixed
permeabilised mouse 3T3 cells in an immunocytochemical assay were selected and
cloned. Hybridoma supernatants were screened against growing and quiescent
3T3 cells in order to identify antibodies against growth-regulated proteins. All the
positive clones were also screened by ELISA to identify, and in the current pro-
11
RNA-binding domain of the 70 K U1 snRNP protein. This region encompasses
the octamer sequence which has been observed in a variety of proteins associated
with RNA (Dreyfuss et al. 1988). In addition, Ro protein contains a potential
“zinebinding finger’’ motif proposed to be involved in nucleic acid-or protein-
protein interactions. The involvement of the putative RNA recognition motif
(RRM) and “zinc finger” motif in binding hY RNA is being determined. Muta-
tions in the RRM and “zinc finger” regions as well as mutations throughout Ro
protein have been created. The recombinant proteins, overexpressed in bacteria or
in vitro translated, have been used in reconstitution assays with hY 1 and hY 3
RNA transcripts in order to define the RNA-binding domain of the 60 kD Ro pro-
tein and to determine the stoichiometry of the complex. Gel shift assays using
mutated Ro translation products and hY 1 RNA have shown that the putative
zinc-finger is not required for binding to hY RNA or DNA. However, the octamer
sequence within the RRM, is required for the interaction of 60 kD Ro with hY
RNA. The sites on the hY RNAs required for interaction with the 60 kD Ro prote-
in are being determined using various mutant hY RNA transcripts reconstituted
with full-length Ro protein. Preliminary studies suggest that sequences at the 3'
end of the hY 1 RNA are not involved in binding Ro protein.
Monoclonal Autoantibodies Against Nuclear Proteins
S. Dyos, L. Alldridge, G. Dealtry, and M. O’Farrell
Department of Biology, University of Essex, Wivenhoe Park, Colchester,
Essex CO4 3SQ, UK
We are investigating the role of nuclear proteins in the control of cell prolifera-
tion. In order to study the structure and function of individual proteins and also
to identify interactions between different proteins we have initiated a programme
to generate monoclonal antibodies to macromolecular components of the
nucleus. We are using autoimmune strains of mice as a biological model system
in which to generate monoclonal antinuclear antibodies. Splenocytes from NZ
B/W Fl female mice (between 4 and 5 months of age) were fused with SP2/0
myelomas . Hybridomas secreting antibodies that recognise the nucleus of fixed
permeabilised mouse 3T3 cells in an immunocytochemical assay were selected and
cloned. Hybridoma supernatants were screened against growing and quiescent
3T3 cells in order to identify antibodies against growth-regulated proteins. All the
positive clones were also screened by ELISA to identify, and in the current pro-