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Human Molecular Genetics Pages 337-343  

The chromosome 15 imprinting centre (IC) region has undergone multiple duplication events and contains an upstream exon of SNRPN that is deleted in all Angelman syndrome patients with an IC microdeletion
Introduction
Results
   Identification of sequences related to the previously identified SNRPN upstream exons
   Methylation status of the [psi]u1C and u1D region
   Expression analysis
   Identification of a novel IC/SNRPN exon in the AS-SRO
Discussion
Materials And Methods
   Patients
   Southern blot hybridization
   Construction of a phage contig for YAC 326F6
   DNA clones and probes
   RT-PCR analysis
   Sequence analysis
Acknowledgements
References

The chromosome 15 imprinting centre (IC) region has undergone multiple duplication events and containsan upstream exon of SNRPN that is deleted in all Angelman syndrome patients with an IC microdeletion

The chromosome 15 imprinting centre (IC) region has undergone multiple duplication events and containsan upstream exon of SNRPN that is deleted in all Angelman syndrome patients with an IC microdeletion

Claudia Färber, Bärbel Dittrich, Karin Buiting and Bernhard Horsthemke*

Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstraße 55, D-45122 Essen, Germany

Received September 23, 1998; Revised and Accepted November 24, 1998

Imprinting of the Prader-Willi/Angelman syndrome region on human chromosome 15 is regulated by an imprinting centre (IC), which spans 5[prime] exons of the gene encoding the small nuclear ribonucleoprotein N (SNRPN). The IC/SNRPN transcripts are initiated at two alternative start sites, which share a high degree of sequence similarity with each other and with two newly identified sites 63 and >700 kb further upstream. Three of these sites are hypermethylated on the maternal chromosome, whereas one displays an oppositemethylation pattern. We have also identified novel splice variants of the IC/SNRPN transcripts and hitherto undetected exons. One of these exons, which we designate u5, is deleted in all Angelman syndromepatients with a microdeletion of the IC. We conclude that elements of the IC region have undergone multiple duplication events and that u5 or a sequence close by may play a role in maternal imprinting.

INTRODUCTION

Proximal 15q contains a 2-3 Mb domain that is subject to genomic imprinting and affected in patients with Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Based on the identification of microdeletions in patients having a wrong imprint on the paternal or the maternal chromosome, respectively, we have suggested that imprinting in 15q is regulated by an imprinting centre (IC) (1). The IC spans the centromeric part of the SNRPN transcription unit and appears to have a bipartite structure (1,2). In PWS families with an imprinting defect, the smallest region of deletion overlap (PWS-SRO) is <4.3 kb and includes the SNRPN CpG island (3,4). These deletions appear to block the maternal to paternal imprint switch in the paternal germline (2). In AS families with an imprinting defect, an ~900 bp region (AS-SRO) immediately distal to an alternative 5[prime] exon of SNRPN called BD3 or IC3 represents the shortest region of deletion overlap (1,2,2; K.Buiting, unpublished data). These deletions appear to block the paternal to maternal imprint switch in the maternal germline (2). As BD3 was deleted in all but one patient, it was unclear whether IC/SNRPN transcripts containing this exon or whether a sequence immediately distal to BD3 was involved in maternal imprinting. We have now investigated the structure and expression of the IC region in detail and have obtained evidence that elements of the IC region are the result of multiple duplication events. Furthermore, we have found a novel 5[prime] exon of SNRPN that is deleted in all AS patients.

RESULTS

Identification of sequences related to the previously identified SNRPN upstream exons

Previous studies (2) identified two alternative 5[prime] exons of the IC/SNRPN transcripts (BD1B and BD1A) and two common exons (BD2 and BD3). Furthermore, some BD1B transcripts were found to contain the alternatively spliced exon BD1B*. For clarity, we will rename the exons u1B (BD1B), u1A (BD1A), u2 (BD2) and u1B* (BD1B*) (for upstream SNRPN exons). Because we have identified another exon between u2 (BD2) and BD3 (see below), BD3 will be renamed u4.

By hybridization of a fetal brain cDNA library with a 625 bp probe containing u1A and flanking sequence (PWCFOA) (2), we have isolated a 520 bp clone containing a sequence similar to u1A and u1B and flanking intronic sequences, suggesting that this clone is of genomic origin rather than a cDNA clone. Comparison of the downstream flanking sequence of this newly identified putative exon with the splice site consensus sequence (7) showed that the exon/intron junction (5[prime] splice site) differs from the consensus sequence in the highly conserved second position of the following intron, thus, this exon is most probably a pseudoexon and we designate it [psi]u1C (Table 1) (GenBank accession no. AF087646). u1B and u1A share a high degree of sequence similarity, whereas [psi]u1C is somewhat less similar to u1A and u1B (Table 2). By hybridization of the [psi]u1C clone to EcoRI-digested YAC clones, which cover part of the PWS/AS region (8,9), a 3.3 kb EcoRI fragment was detected in YAC 71B11 (data not shown). YAC 71B11 contains the anonymous marker D15S11, which maps >700 kb centromeric to SNRPN (Fig. 1).


Table 1. Splice site sequences of SNRPN upstream exons
Numbers given for the consensus sequence nucleotides indicate percentage occurrence. Highly conserved nucleotides (100%) are shown in bold. Exon sequences are indicated by upper case letters, and flanking intron sequences by lower case letters. Splice sites for u1A, u1B, u1B*, u2 and u4 have been published before (2).

To address the question whether 15q11-q13 contains additional sequences related to u1B, u1A and [psi]u1C, we hybridized the same YACs with PWCFOA (2). By this we detected two EcoRI fragments known to contain u1A (4.7 kb) and u1B (9.6 kb) in YAC 326F6, as well as one more EcoRI fragment (7.2 kb) in YAC 326F6 and also in the overlapping YAC 307A12 (data not shown). To isolate this cross-hybridizing sequence, YAC 326F6 was subcloned into [lambda] phage clones and, by walking in this library, we extended our previously established phage contig containing u1B, u1A and SNRPN (1,2) by 75 kb (Fig. 1). Partial sequence analysis of the 7.2 kb cross-hybridizing EcoRI fragment revealed a sequence closely related to u1A and u1B (hence named u1D; GenBank accession no. AF087645) (Table 2). Analysis of the downstream sequence of u1D showed that the putative exon/intron boundary of u1D matches the splice site consensus sequence (Table 1). Based on the restriction map, u1D maps ~63 kb centromeric to u1B (Fig. 1). PCR analysis with specific primers for u1D and primers annealing to adjacent EcoRI phage fragments demonstrated that u1D has the same 5[prime]->3[prime] orientation on the chromosome as u1A and u1B (data not shown). A search of the NCBI database with exons u1A and u1B identified a 132 kb genomic sequence (GenBank accession no. AC004737), which contains u1A and u1B, but not u1D. Comparison of this sequence with a 2.2 kb sequence spanning u1D revealed ~76% sequence similarity to sequences around u1A and u1B over >1.6 kb, suggesting that not only the exons, but larger blocks of DNA have been duplicated.


Figure 1. Order of the [psi]u1C, u1D, u1B (previously named BD1B) and u1A (previously named BD1A) regions in proximal 15q11 (top). The relative orientation of [psi]u1C and D15S11 and the precise distances of [psi]u1C and D15S11 to D15S13 are not known. The EcoRI (E) restriction map of the IC/SNRPN region is shown in the lower part. The phage contig links u1D to the IC/SNRPN transcription unit. Phage clones designated [lambda]45.x are subclones of YAC 326F6. Phage clones [lambda][alpha]39, [lambda]71.x and [lambda]48.x are part of a previously described contig (1,2). Black boxes represent exons 1-10 of the SNRPN gene and 5[prime] exons of SNRPN: u1B (BD1B), u1B* (BD1B*), u1A (BD1A), [psi]u1C, u1D, [psi]u1D* and [psi]u1A*. The open circles represent anonymous markers.

Table 2. Sequence similarities of SNRPN upstream exons (%)
Exon u1A u1B [psi]u1C
u1A      
u1B 84.3    
[psi]u1C 70.6 67.4  
u1D 85.7 78.9 65.4

By hybridization of the phage contig with a probe for the alternatively spliced exon u1B*, we identified two homologous sequences: one ~35 kb downstream of u1D and the other ~6.5 kb downstream of u1A. As the downstream sequences of these putative exons do not match the exon/intron consensus sequence (7), these exons are most probably pseudoexons and were therefore named [psi]u1D* (GenBank accession no. AF087647) and [psi]u1A* (GenBank accession no. AF087648) (Fig. 1 and Table 1).


[psi]u1D*, [psi]u1A* and u1B* share a high degree of sequence similarity (Table ). Analysis of the flanking DNA and homology searches with the sequence AC004737 showed that sequences around [psi]u1D*, u1B* and [psi]u1A* have a sequence similarity of ~71% over at least 900 bp. Interestingly, the genomic distances between u1B and u1B* (5.9 kb) and u1A and [psi]u1A* (6.5 kb) are rather similar, but the distance between u1D and [psi]u1D* is >35 kb (Fig. 1). Hybridization of YAC clones from the region around [psi]u1C with the same probe did not show any evidence for a related sequence near [psi]u1C.

Table 3. Sequence similarities of SNRPN upstream exons (%)
Exon [psi]u1A* u1B*
[psi]u1A*    
u1B* 80.8  
[psi]u1D* 81.8 75.9

Methylation status of the [psi]u1C and u1D region

As the u1B and u1A regions are methylated on the maternal chromosome and unmethylated on the paternal chromosome (2), we determined the methylation status of [psi]u1C and u1D. PCR-generated probes were hybridized to HindIII+HpaII- or HindIII+CfoI-digested DNA from normal individuals, PWS patients and AS patients.

For [psi]u1C we identified a differentially methylated HpaII site (Fig. 2). Normal individuals have an upper band that is more intense than the lower band. PWS patients, who lack the paternal copy of 15q11-q13, have the upper band only. AS patients, who lack the maternal copy of 15q11-q13, have an upper band of reduced intensity compared with normal individuals. We conclude that the HpaII site is methylated on the maternal chromosome, whereas the paternal allele is unmethylated in ~50% of the cells.


Figure 2. Parent-of-origin-specific methylation patterns. Genomic DNA from normal individuals, PWS patients and AS patients was digested with HindIII+HpaII or HindIII+CfoI and hybridized with PW71B (u1A region) (16), the subclone pa30.12 (u1B region) (2), the 520 bp [psi]u1C clone ([psi]u1C region) and with a PCR-generated hybridization probe for the u1D region. mat, maternal; pat, paternal.


Figure 3. RT-PCR analysis. Human adult testis RNA was reverse transcribed and amplified (a) using primers specific for u1B (B48x) and SNRPN exon 2 (B62SmNO) (473 bp, RT1; 436 bp, RT2; 413 bp, RT3; 380 bp, RT4; 339 bp, RT5; 305 bp, RT6; 225 bp, RT7; 191 bp, RT8; see also Fig. 4) and (b) using primers for u1A (KB74a) and SNRPN exon 2 (B62SmNO) (478 bp, RT10; 441 bp, RT11; 426 bp, RT12; 385 bp, RT13; 370 bp, which could not be identified by reamplification; 310 bp, RT14; 236 bp, RT15; 122 bp, RT17). M, 1 kb ladder (Life Technologies, Eggenstein, Germany).

For u1D we detected a CfoI site with an opposite methylation pattern (Fig. 2). PWS patients have a strongly reduced upper band compared with normal individuals and in AS patients the lower band is much fainter than the upper band. Therefore, the CfoI site is hypomethylated on the maternal chromosome and hypermethylated on the paternal chromosome. Mapping studies indicate that this site lies ~8.2 kb distal of u1D (Fig. 1). This is in contrast to the differentially methylated CfoI sites near u1A and u1B, which map ~50 bp upstream of these exons (2).

Expression analysis

To investigate whether u1D functions as an additional start site for the IC/SNRPN transcripts, we performed exon-connecting RT-PCR with a specific primer for u1D (RTD6) in combination with a primer annealing to [psi]u1D* (pdst1), exon u2 (B48r), exon u4 (B48t) and SNRPN exon 2 (B62SmNO), respectively. The RNA was from adult brain, adult testis, fetal lung and fetal brain, which are known to contain IC/SNRPN transcripts (2). This analysis failed to reveal specific RT-PCR products in each case (data not shown). Similar experiments with a primer specific for u1A (KB74a) and a primer for [psi]u1A* (past1) also failed (data not shown), in agreement with the assumption that [psi]u1A* is a pseudoexon.

RT-PCR experiments with adult testis RNA using primers specific for u1B (B48x) and u1A (KB74a) in combination with a primer for SNRPN exon 2 (B62SmNO) revealed a major u1B RT-PCR product of 305 bp (RT6) and less prominent u1B RT-PCR products of 473 (RT1), 436 (RT2), 413 (RT3), 380 (RT4), 339 (RT5), 225 (RT7) and 191 bp (RT8) (Figs 3a and 4). Furthermore, we obtained a major u1A RT-PCR product (310 bp, RT14) and other u1A RT-PCR products of 478 (RT10), 441 (RT11), 426 (RT12), 385 (RT13), 370, 236 (RT15) and 122 bp (RT17) (Figs 3b and 4). A similar pattern of u1A and u1B transcripts was obtained in fetal brain RNA (data not shown). Reamplification of RT-PCR products from fetal brain displayed one more product (117 bp) initiated at u1B (RT9) and one more product (196 bp) initiated at u1A (RT16) (data not shown). Sequence analysis of all RT-PCR products revealed that RT3, RT6 and RT14 correspond to the previously identified RT-PCR products amplified with a primer specific for u1B or u1A in combination with a primer for u4 (2). Other RT-PCR products (RT5, RT7-9 and RT15-17) were identified as new splice variants of the IC/SNRPN transcripts resulting from alternative splicing (Fig. 4). In summary, we identified five new variants of the IC/SNRPN transcripts lacking exon u2 (RT5, RT7, RT9, RT15 and RT17) and five alternative transcripts lacking exon u4 (RT7-9, RT16 and RT17) (Fig. 4).


Figure 4. Overview of the IC/SNRPN transcription unit (not drawn to scale). Transcription start sites are indicated by arrows. The question mark indicates unclear transcriptional activity of u1D; [psi], pseudoexons. The relative position of the differentially methylated HpaII site near [psi]u1C is not known. Lollipops, CpG dinucleotides; mat, maternal chromosome; pat, paternal chromosome; grey boxes, IC/SNRPN exons (u); hatched boxes, Alu exon sequences; black boxes, open reading frame of SNRPN. RT, RT-PCR products (amplified with primers for u1B or u1A and SNRPN exon 2 from adult testis RNA (RT1, RT2, RT5-8, RT10, RT12, RT14 and RT15) and fetal brain RNA (RT3, RT4, RT9, RT11, RT13, RT16 and RT17) representing splice variants of the IC/SNRPN transcripts.

In one u1B RT-PCR product (473 bp, RT1) as well as in one u1A RT-PCR product (478 bp, RT10), both isolated from adult testis RNA, a new exon of 168 bp (named u3; GenBank accession no. AF092911) was identified between u2 and u4 (Fig. 4). This exon was mapped 7.5 kb upstream of u4 (Fig. 1) and sequence analysis showed conserved splice site sequences for this newly identified exon (Table 1).

In two identical RT-PCR subclones initiated at u1A (RT12) and isolated from fetal brain RNA and adult testis RNA, respectively, we identified a 116 bp sequence homologous to the Alu sequence family. This sequence maps ~4.3 kb proximal to SNRPN exon 1 (Fig. 4). In one other u1A RT-PCR product from fetal brain RNA (RT11) as well as in one u1B RT-PCR product from adult testis (RT2), we obtained a 131 bp exon sequence which is also homologous to the Alu sequence family. This sequence maps 3.6 kb distal of SNRPN exon 1 and ~3 kb downstream of the distal breakpoint of family PWS-O, the current distal border of the PWS-SRO (1,3,4). Interestingly, homology search with this exon revealed an EST clone from fetal brain (GenBank accession no. T03430), which contains the identical Alu sequence spliced between SNRPN exons 1 and 2 (10). Similarly, an EST clone from adult brain (GenBank accession no. H30300) contains a 102 bp Alu sequence between exons u4 and SNRPN exon 2. This sequence maps ~15 kb proximal to SNRPN exon 1. All three Alu exon sequences match the consensus splice site sequences (data not shown).

Identification of a novel IC/SNRPN exon in the AS-SRO

In four identical RT-PCR subclones initiated at u1B (380 bp, RT4) and in four subcloned RT-PCR products initiated at u1A (385 bp, RT13), all isolated from fetal brain RNA, another novel exon of 75 bp was identified. This exon shows sequence identity to a sequence mapping 342 bp downstream of u4 and was therefore named u5 (GenBank accession no. AF087649) (Figs 1 and 4). The genomic sequence around u5 showed that this exon has conserved splice sites (Table 1). The proximal border of the smallest region of deletion overlap in AS patients with an imprinting defect (AS-SRO) is represented by the breakpoint of family AS-H, which lies 288 bp distal to exon u4 (2). Thus, u5 maps 54 bp distal to the breakpoint of AS-H within the AS-SRO.

DISCUSSION

In a comprehensive analysis of the structure and coding potential of the SNRPN upstream region we have found: (i) that elements of the human chromosome 15 IC region have undergone multiple duplication events; (ii) that the IC/SNRPN transcripts occur in multiple alternatively spliced forms; and (iii) that a hitherto undetected exon is deleted in all AS patients with an IC deletion.

(i) Previous studies (2) had revealed that the IC/SNRPN transcripts are initiated at two alternative start sites (the u1A and the u1B region), which share a high degree of sequence similarity and which are unmethylated on the paternal chromosome, but methylated on the maternal chromosome. The methylation pattern was in agreement with the finding that u1A- and u1B-containing transcripts are made from the paternal chromosome only. Here we report that there are two more related sequences further upstream, one of which (the u1D region) has an opposite methylation pattern. Although the differentially methylated CpG dinucleotides are not conserved, our finding suggested that there might be additional and possibly maternally expressed IC/SNRPN transcripts. However, we have not obtained any evidence for the presence of such transcripts in adult brain, adult testis, fetal brain or fetal lung, which are known to contain IC/SNRPN transcripts. It is possible that u1D is expressed in tissues other than those containing transcripts initiated at u1A or u1B or that it is not expressed at all. The distance between u1D and the downstream exon [psi]u1D* is much bigger compared with the distance between u1B and u1B*, for example, suggesting that an insertion leading to the silencing of u1D might have occurred during evolution. As [psi]u1C, which maps >700 kb upstream of u1B, does not have a conserved exon/intron junction and is most likely a pseudoexon, we did not expect transcripts initiated at this locus.

The duplication also includes exons u1B*, [psi]u1D* and [psi]u1A*, whereas we have not obtained any evidence for such a sequence in the [psi]u1C region by Southern blot analysis. The presence of duplicated exons may be explained by the following evolutionary scenario. An ancestral sequence including one start exon and one other exon or pseudoexon was duplicated to yield u1B/u1B* and u1A/[psi]u1A*. Another duplication followed by an insertion then gave rise to u1D/[psi]u1D*. Finally, a third duplication event led to [psi]u1C. As the duplications do not only include the exons, but flanking DNA as well, the duplications are probably not the result of retrotranspositions, but of chromosomal events such as unequal crossovers. We can only speculate about the functional significance of these duplications. The presence of multiple start sites (at least u1A and u1B) may ensure expression of the IC/SNRPN transcripts even in the event of a deletion of one site, may provide functional divergence or may just represent evolutionary noise.

(ii) By extensive RT-PCR analysis, we have found evidence for the presence of almost all possible alternative splice forms, at least in adult testis and fetal brain (Fig. 4). At first glance, the finding of transcripts lacking u2 may cast doubt on the functional significance of a putative splice site mutation found in family AS-C2 predicted to lead to skipping of this exon (2). However, an individual with this mutation will not make any u2-containing transcript, whereas in other individuals transcripts with and without u2 occur. As we do not know if the IC/SNRPN transcripts really have a function, the significance of the u2 mutation in family AS-C2 remains unclear. As an alternative to a role of the transcripts in imprint switching in the maternal germline (2), it is also possible that the transcripts per se are irrelevant for this process, but that transcription through the IC/SNRPN locus is important, as has been suggested for the H19 region (11). In this scenario, the primary transcript is just spliced in almost all possible forms. A point in case is the finding of rare transcripts containing splice fragments of Alu sequences. Although the immediate splice sites of the u exons and the ‘Alu exons’ match the consensus sequences, the splice sites may be weak and predispose to exon skipping.

(iii) Most interestingly, we have identified a novel SNRPN upstream exon (u5), which is deleted in all AS patients with an IC deletion. Previous studies had revealed that all but one patient (AS-H) had a deletion including exon u4 (BD3). In AS-H, the deletion started 288 bp distal to u4 and the deletion of regulatory elements downstream of u4 had to be invoked to explain a possible effect of the AS-H deletion on expression of the IC/SNRPN transcripts (2). All patients including AS-H are deleted for at least one SNRPN upstream exon (u5). Although it may be argued that the deletion of u5 is irrelevant, because several splice forms lack this exon, individuals having a constitutional deletion will not make any u5-containing transcript at all (see above discussion about the u2 splice site mutation) and this transcript or the mixture of all splice forms may be relevant for imprint switching. Furthermore, the splicing pattern of the IC/SNRPN transcripts in primordial germ cells may be different from that in fetal brain and adult testis. However, RNA from these cells is not available for testing. As recently suggested (11), the IC/SNRPN transcripts may be involved in the assembly of an imprint initiation complex in the maternal germline. Thus, assembly of the imprint initiation complex might be dependent on the presence of transcripts containing u5.

Of course, we are well aware of alternative explanations. The shortest region of deletion overlap in AS patients (AS-SRO) has recently been narrowed to ~900 bp (12; K.Buiting, unpublished data) and may not only contain u5, but an as yet unidentified regulatory element as well. It may be this element that is important in cis for maternal imprinting and its close proximity to u5 may be fortuitous. This might explain why we have not obtained any evidence so far for point mutations in the AS-SRO including u5 in non-IC deletion patients with an imprinting defect (12). Unlike the H19 upstream region, however, which is important for imprinting of the Igf2 and H19 genes (for a review see ref. 12), the AS-SRO is not differentially methylated (14) and thus unlikely to bear the primary epigenetic mark from which the imprints spread. In contrast, there is good evidence from mouse and patient studies that differential methylation of the SNRPN CpG island represents the primary epigenetic mark in this region (15).

What then is the role of the AS-SRO in maternal imprinting? As discussed above, transcription through the SNRPN upstream region may be important and this activity may be regulated by a factor binding to the AS-SRO. Alternatively, this region contains an as yet unknown DNA element which exerts some effect independent of the IC/SNRPN transcription, for example by interfering with the switch initiation site. The finding of a novel exon in the AS-SRO, however, appears to strengthen the case for a role of the IC/SNRPN transcripts in imprint switching, although further experiments will have to address these questions.

MATERIALS AND METHODS

Patients

All patients studied had typical AS or PWS as confirmed by methylation analysis of D15S63 and SNRPN.

Southern blot hybridization

Genomic DNA and phage DNA were isolated by standard methods (16). Aliquots of the DNA were digested with the appropriate restriction enzymes. Fragments were separated by agarose gel electrophoresis, transferred to Biodyne A nylon membrane (Pall, Portsmouth, UK) and hybridized with 32P-labelled probes as described (17). The final wash was in 2× SSC, 0.1% SDS at 65°C for 20 min.

Construction of a phage contig for YAC 326F6

Yeast DNA (50 µg) containing YAC 326F6 was partially digested with Sau3AI and subcloned as described (16). To identify clones with human insert DNA, a total of 9000 plaques were hybridized with radioactively labelled human genomic DNA. Positive plaques (122) were hybridized with 32P-labelled phage inserts or with specific probes. Phage DNA was digested with EcoRI, separated on 0.8% agarose gels, transferred to Biodyne A nylon membrane (Pall) and hybridized with anchor probes and putative phage end fragments to construct the EcoRI restriction map.

DNA clones and probes

The following YAC clones were used for the identification of putative additional start sites of the IC transcripts: 71B11, 307A12, 326F6, B58C7 and 457B4 (8,9). YACs were hybridized with a PCR probe PWCFOA (containing u1A and flanking sequences) amplified with primers PWCFO1/2 (5[prime]-GAATGCGAACATGCGAAG-3[prime] and 5[prime]-CTCTATGCCTTGAACCTACACC-3[prime], annealing temperature 58°C) using phage clone [lambda]71.13 as a template. For methylation analysis of the u1B region, the plasmid subclone pa30.12 was used (2). The CfoI site in the u1A region is detected by the probe PW71B (16). For methylation analysis of the [psi]u1C region, the 520 bp [psi]u1C clone was used and for the u1D region a hybridization probe was generated by PCR amplification with the consensus sequence primers PWCFO3/4 (5[prime]-AGACCCCACAGAAGGCTCTG-3[prime] and 5[prime]-GTCTTTAATGTTCTTGTGAATC-3[prime], annealing temperature 60°C) using YAC 326F6 as a template. The hybridization probe for u1B* was amplified from genomic DNA using primers BDex1vr/s (5[prime]-GTTGGTGCTGAGGACAAAAG-3[prime] and 5[prime]-GTGGTCATGCACGTACACTG-3[prime], annealing temperature 58°C). A probe for exon u5 was generated by PCR using primers u5exr/s (5[prime]-CCTCCTCAGATTTGGCACA-3[prime] and 5[prime]-ACTTGAGTGTGCATTAGTATC-3[prime], annealing temperature 58°C) with the phage clone [lambda]48.8 as a template.

RT-PCR analysis

RT-PCRs were performed with the GeneAmp RNA PCR Kit (Perkin Elmer, Foster City, CA). A total of 150 ng of RNA from fetal brain, fetal lung and adult brain was reverse transcribed using random hexamers in a final volume of 20 µl, both in the presence and in the absence of reverse transcriptase (RNA + RT and RNA - RT), to control for contaminating DNA sequences. The resulting cDNA products were amplified in 100 µl volume by 35 cycles using the following primers and annealing temperatures: RTD6 (5[prime]-CAGCTGTCCCCTGGGATA-3[prime]) + B62SmNO (2) (annealing temperature 60°C); RTD6 + B48r (2) (annealing temperature 58°C); RTD6 + B48t (2) (annealing temperature 58°C); RTD6 + u5r (5[prime]-CTCTCAACGTGTGTTCCTC-3[prime], annealing temperature 56°C); RTD6 + pdst1 (5[prime]-TGTAGTGTATTGATACCAGGC-3[prime], annealing temperature 58°C); B48x (2) + B62SmNO (annealing temperature 60°C); KB74a (2) + past1 (5[prime]-ACCCTGTGATGCAAGGTGG-3[prime], annealing temperature 60°C); KB74a + B62SmNO (annealing temperature 60°C). RT-PCR products were reamplified, subcloned into a dT-tailed pBluescript vector and sequenced.

Sequence analysis

Plasmid DNA was sequenced with vector-specific primers (M13r and M13s), fluorescence-tagged dideoxynucleotides and the Taq cycle sequencing procedure (ABI, Foster City, CA). PCR products were purified with Microcon-100 microconcentrators (Amicon, Beverley, MA) and analysed on an ABI DNA Sequencer 377A. Sequence similarities were investigated using the Jotun Hein method, Lasergene software (DNAStar, Madison, WI) and by searching the NCBI databank.

ACKNOWLEDGEMENTS

We thank Dr V. Kalscheuer (Berlin) and S. Endele (Mainz) for fetal RNA samples and S. Groß and C. Lich for expert sequencing and technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and the Human Frontier Science Program Organization.

REFERENCES

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*To whom correspondence should be addressed. Tel: +49 201 7234556; Fax: +49 201 7235900; Email: b.horsthemke@uni-essen.de

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