DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.48120#0026
8
Molecular and Cell Biology of Autoantibodies and Autoimmunity
lecular weight of approximately 140 kD. These fusion proteins showed positive
reactions on Western blots with murine monoclonal and human polyclonal anti-
B/B' antibodies. Human antibodies eluted from a blot containing fusion protein
were shown to react strongly with B/B' on a HeLa nuclear blot. In addition, a
weaker reaction of these antibodies with the A protein was observed. Clone 16
contained a cDNA insert of 914 nucleotides, containing an open reading frame
encoding 252 amino acids. If the first ATG codon, which occurs in an nearby op-
timal context for translation initiation, is regarded as the initiation site for transla-
tion, a 240 amino acid protein is encoded.
From clone 5 a cDNA insert of 1049 nucleotides was isolated. This clone
started 15 nucleotides more upstream than clone 16. Subsequently, 719 nucleo-
tides were identical in both clones. At this position, clone 5 contained an insert
of 146 nucleotides, introducing a termination codon in the coding region.
Therefore, clone 5 encodes a 231 amino acid protein. A striking feature of the in-
sert was the excellent homology with the consensus sequence for the 3'-intron ac-
ceptor junction for pre-mRNA splicing.
Clone 9 started at the 5' end at the same nucleotide as clone 5 and was iden-
tical to clone 5 apart from one non-silent mutation at position 359. An additional
screening of a human cDNA placenta library, using a random-primed cDNA in-
sert of clone 16, resulted in 24 positive clones, of which 8 clones also hybridized
with a synthetic oligonucleotide complementary to the 5' end of clone 16. Restric-
tion analysis of four of these clones showed that two clones comprised the 146
nucleotide insert, whereas the two other clones lacked it.
In vitro transcription and translation of cDNAs showed that clone 5 encoded
a protein with a molecular weight of 29.5 kD on SDS-gels, whereas clone 16 en-
coded a 28.5 kD protein. Both proteins were immunoprecipitable with an anti-Sm
serum and showed a difference of approximately 0.5 kD if compared with labelled
HeLa snRNP proteins B' and B (29 and 28 kD).
To confirm that the two mRNAs corresponding with clone 5 and 16 were both
present in human cells, an RNase mapping experiment was performed.
Nucleotide fragments 665-899 from clone 5 and 650-738 from clone 16, obtain-
ed by restriction enzyme digestion, were used as probes. These fragments spanned
the region containing the additional insert. The fragments from HeLa and HL-60
RNA which were protected were 233 and 73 nucleotides if the cDNA fragment
from clone 5 was used as a probe, whereas the fragment from clone 16 protected
a 87 nucleotide fragment. These fragments correspond with the fragments we
should expect to be protected by the respective probes, if both the short and the
long mRNA species were present in cytoplasmic mRNA.
From these results we conclude that B and B' differ only in their carboxy-ter-
minal part, where B' contains one more repeat of a proline-rich motive. The pro-
teins are encoded by two distinct mRNAs, which are most probably generated
from one common pre-mRNA by alternative splicing. Both proteins are extremely
proline-rich, especially at their carboxyterminal part. Homologies with the pub-
Molecular and Cell Biology of Autoantibodies and Autoimmunity
lecular weight of approximately 140 kD. These fusion proteins showed positive
reactions on Western blots with murine monoclonal and human polyclonal anti-
B/B' antibodies. Human antibodies eluted from a blot containing fusion protein
were shown to react strongly with B/B' on a HeLa nuclear blot. In addition, a
weaker reaction of these antibodies with the A protein was observed. Clone 16
contained a cDNA insert of 914 nucleotides, containing an open reading frame
encoding 252 amino acids. If the first ATG codon, which occurs in an nearby op-
timal context for translation initiation, is regarded as the initiation site for transla-
tion, a 240 amino acid protein is encoded.
From clone 5 a cDNA insert of 1049 nucleotides was isolated. This clone
started 15 nucleotides more upstream than clone 16. Subsequently, 719 nucleo-
tides were identical in both clones. At this position, clone 5 contained an insert
of 146 nucleotides, introducing a termination codon in the coding region.
Therefore, clone 5 encodes a 231 amino acid protein. A striking feature of the in-
sert was the excellent homology with the consensus sequence for the 3'-intron ac-
ceptor junction for pre-mRNA splicing.
Clone 9 started at the 5' end at the same nucleotide as clone 5 and was iden-
tical to clone 5 apart from one non-silent mutation at position 359. An additional
screening of a human cDNA placenta library, using a random-primed cDNA in-
sert of clone 16, resulted in 24 positive clones, of which 8 clones also hybridized
with a synthetic oligonucleotide complementary to the 5' end of clone 16. Restric-
tion analysis of four of these clones showed that two clones comprised the 146
nucleotide insert, whereas the two other clones lacked it.
In vitro transcription and translation of cDNAs showed that clone 5 encoded
a protein with a molecular weight of 29.5 kD on SDS-gels, whereas clone 16 en-
coded a 28.5 kD protein. Both proteins were immunoprecipitable with an anti-Sm
serum and showed a difference of approximately 0.5 kD if compared with labelled
HeLa snRNP proteins B' and B (29 and 28 kD).
To confirm that the two mRNAs corresponding with clone 5 and 16 were both
present in human cells, an RNase mapping experiment was performed.
Nucleotide fragments 665-899 from clone 5 and 650-738 from clone 16, obtain-
ed by restriction enzyme digestion, were used as probes. These fragments spanned
the region containing the additional insert. The fragments from HeLa and HL-60
RNA which were protected were 233 and 73 nucleotides if the cDNA fragment
from clone 5 was used as a probe, whereas the fragment from clone 16 protected
a 87 nucleotide fragment. These fragments correspond with the fragments we
should expect to be protected by the respective probes, if both the short and the
long mRNA species were present in cytoplasmic mRNA.
From these results we conclude that B and B' differ only in their carboxy-ter-
minal part, where B' contains one more repeat of a proline-rich motive. The pro-
teins are encoded by two distinct mRNAs, which are most probably generated
from one common pre-mRNA by alternative splicing. Both proteins are extremely
proline-rich, especially at their carboxyterminal part. Homologies with the pub-