Plant Molecular Biology 45: 449–460, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Differential expression of potato U1A spliceosomal protein genes: a rapid
method for expression proﬁling of multigene families
Adel F.M. Ibrahim, Jenny A. Watters and John W.S. Brown
Department of Cell & Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA,
Scotland, UK (
author for correspondence; e-mail: email@example.com)
Received 20 December 1999; accepted in revised form 19 October 2000
Key words: expression proﬁling, multigene family, 5
The spliceosomal protein, U1A, is a component of the U1snRNP essential to pre-mRNA splicing. From the
ubiquitous nature of the splicing machinery, expression of U1A genes is expected to be constitutive. However,
many plant genes are organised in multigene families that exhibit variation in expression proﬁles. Without detailed
knowledge of the size of the U1A gene family or their degree of sequence variation, we examined the expression
of the U1A genes using a novel approach. The approach was based on 5
RACE with [
P]-labelled primers and
separationofproductson high-resolutionDNA sequencinggelstogive a ‘snapshot’of theexpression ofU1A genes.
This was followed by sequencing of cloned 5
RACE productsand of products re-ampliﬁed from excised bands. In
combination with RT-PCR/SSCP, these analyses allowed the rapid identiﬁcation of different gene transcripts and
assessment of their relative expression proﬁles. Transcriptsfrom four U1A genes (U1A-1 to U1A-4) were identiﬁed,
of which U1A-1 and U1A-2 were expressed much more highly than U1A-3 and U1A-4. Differential expression of
the twomosthighlyexpressedgenes, U1A-1 and U1A-2, was observedin that onlyU1A-2 was expressed in ﬂowers.
Upstream sequences of U1A-1 and U1A-2 were cloned and the gene-speciﬁc promoters identiﬁed on the basis of
the sequence variation deﬁned from the 5
RACE products. The differential expression of these genes may be due
to a 1.3 kb insertion less than 200 bp upstream of the U1A-1 coding sequence. This approach can be used more
generally to examine expression proﬁles of multigene families, and in particular to reﬁne information from EST or
microarray analyses, and to isolate rapidly gene-speciﬁc promoters.
Removal of introns from precursor messenger RNA
(pre-mRNA splicing) is a fundamentalprocess in gene
expression and one level at which expression can be
regulated. Splicing occurs in a large ribonucleopro-
tein complex called the spliceosome (Moore et al.,
1993; Krämer, 1995). The major components of the
spliceosome are four small nuclear ribonucleoprotein
particles (snRNPs): U1, U2, U4/U6 and U5. Each
snRNP contains one or two small nuclear RNAs (snR-
NAs), a set of core proteins common to each snRNP,
and snRNP-speciﬁc proteins. For example, U1snRNP
contains three U1snRNP-speciﬁc proteins: U1A, U1C
and U1-70K. Spliceosomal snRNPs are abundant in
higher eukaryotic cells; in human cells there are ca.
U1snRNPs, 5 × 10
U2snRNPs and 2 × 10
U4/U6 and U5snRNPs (Reddy and Busch, 1982).
The spliceosome also contains numerous transiently
associated spliceosomal proteins. The housekeeping
functionof splicing and the abundanceof snRNPs sug-
gest that many snRNP and spliceosomal proteins will
be constitutively expressed at relatively high levels.
Although the majority of snRNP proteins have
been identiﬁed in yeast and man, and genes for many
of them have been cloned, very few genes for plant
snRNP proteins have been characterised. Genes for
core proteins have been identiﬁed (Seraphin, 1995),
and genes for U1snRNP-speciﬁc proteins, U1A and
U1-70K, the U2snRNP-speciﬁc protein, U2B
the U5snRNP-speciﬁc protein PRP8 have been iso-
lated (Simpson et al., 1991, 1995; Golovkin and