rrohan at world.std.com
Mon Sep 11 05:40:01 EST 1995
jon boyson <boyson at primate.wisc.edu> wrote:
>I need references and help from people familiar with alternative
>splicing. I've isolated alternatively spliced cDNAs from a MHC class I
>gene which appear fairly "normal," i.e., they are spliced exon to exon.
>However, I've also isolated a fair number of cDNAs from the same prep
>that are spliced in a rather haphazard fashion, e.g., the middle of one
>exon is spliced to the 3'UTR, others have deletions near intron/exon
>As our lab has little experience in studying splicing, I want to know if
>these are "real," or are they splicing intermediates, or could they be
>experimental artifact (these were all subcloned from RT/PCR reactions
>from total RNA)? Would we see any difference if we isolate message
>only? Does anyone have an excellent reference on the subject?
Using RT-PCR, Aatsinki et al. isolated multiple alternative splice variants of
rat LH/hCG receptor, many of which do not use the "normal" intron/exon
boundaries (Mol Cell Endocrinol 1992 84:127-135). I know of at least one
instance in which a variant RNA was isolated by RT-PCR but could not be
confirmed by RNase protection assay (Zorn AM. Krieg PA. PCR analysis of
alternative splicing pathways: identification of artifacts generated by
heteroduplex formation. Biotechniques. 11(2):180, 182, 184, 1991 Aug). It is
also possible that you have amplified splicing errors that constitute a small
fraction of the MHC class I mRNA. The protection assay would confirm the
presence of your variants within the total RNA population and also document
the relative abundance of each type (in the spirit of blatant self promotion
see DJ Bacich et al Endocrinology 135: 735-744, 1994). A good, but somewhat
old, review on splicing was done by CWJ Smith et al (Annu Rev Genet
23:527-577, 1989). A more recent literature survey has turned up the
Inoue K. Ohno M. Shimura Y. Aspects of splice site selection in constitutive
and alternative pre-mRNA splicing. Gene Expression. 4:177-82, 1995.
Horowitz DS. Krainer AR. Mechanisms for selecting 5' splice sites in mammalian
pre-mRNA splicing. Trends in Genetics. 10:100-6, 1994.
Norton PA. Alternative pre-mRNA splicing: factors involved in splice site
selection. Journal of Cell Science. 107:1-7, 1994.
Hodges D. Bernstein SI. Genetic and biochemical analysis of alternative RNA
splicing. Advances in Genetics. 31:207-81, 1994.
Helfman DM. The generation of protein isoform diversity by alternative RNA
splicing. Society of General Physiologists Series. 9:105-15, 1994.
Rich Rohan, PhD (rrohan at world.std.com)
Independent Molecular Biology Consultant to the Internet
Moderator REPRENDO at world.std.com - Reproductive Endocrinology List
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