Plant Molecular Biology 19: 1045-1047, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
1045
Update section Sequence
Nucleotide sequence of the 5S rRNA gene from
Glycine soja
Alexander Kolchinsky and Peter M. Gresshoff Plant Molecular Genetics and Center for Legume Research, University of Tennessee, Knoxville, TN 37901-1071, USA Received 5 March 1992; accepted in revised form 23 March 1992
Key words: soybean (Glycine sp.), non-transcribed spacer, PCR
The non-transcribed spacer (NTS) of the 5S gene from Glycine soja (Sieb and Zucc., PI468.397) was amplified using the polymerase chain reaction (PCR) with primers universal for plant nuclear 5S genes described earlier [6] The primers are complementary to the 3' and 5' ends of highly conservative coding region and amplify divergent NTS sequences. The amplified fragment was cloned into the pCR1000 vector (Invitrogen) designed for direct cloning of PCR products. The vector has hanging 3'-thymidine residues complementary to adenine residues added to the 5' ends of PCR products by Taq-polymerase. Both strands of the cloned NTS fragment were sequenced using the TaqTrack dideoxy-kit (Promega). The resulting nucleotide sequence for the G. soja 5S rRNA gene is shown in Fig. 1. The length of the cloned PCR product was 255 bp, the primers are 20 bp long each, therefore the presumed length of the NTS was 215 bp, and the whole repeat was 335 bp long. These estimates correspond to the length of initial PCR products used for cloning and their oligomers (not shown). The role of the 5'-flanking region in the transcription of 5S rRNA gene by RNA-poly-
merase III is not quite clear. In contrast to earlier suggestions (see [2]), more recent data indicate that the promoter is not confined to the coding region and involves 5'-flanking sequences [7]. The analysis of the NTS of G. soja sequenced in this work, and its comparison to 5' regions from other plants [3, 4], showed clear homology to putative promoters (Fig. 2). They contained a consensus 'TATA' sequence 29 bp upstream from beginning of transcription, found to be required for in vitro transcription in some species [7]. The sequences surrounding the start of transcription in these cases were also similar. However, some 5S sequences (for example, the barley gene [5]) did not contain any obvious TATA-Iike sequences at proper positions (see compilation in [9]). The transcription termination signal for RNApolymerase III is thought to be a run of 4-5 thymidines surrounded on both sides with 2-3 GC pairs [1]. The sequenced 3'-downstream region of the G. soja 5S rRNA gene contained a typical run of 5 thymidines with CC and CG sequences in the close vicinity on both sides. An unusually long run of 17 thymidine residues followed this termination signal. Southern hybridization of G. soja and soybean
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X64631.
1046 tTGGGAAGTCC.TCGTGTTGCA.CCTCTTTTTA.CGTTTTTTTT.TTTTTTTTTC.
50
TTTTTGCCCT.TATTCTGAGT.ATTTTTCTTT.GAAGCGAAGT.AAAAGGTCGA.
i00
TAAGTTAACT.AATTTTTGTG.ATTGATCGGG.AGATAAATGT.ATCGTGGCCC.
150
GTGCTCGTTC.GTGTAGAAGG.TCGATCGAAA.GTCGCCGTCC.GGCAGCAGAA.
200
GGAATATAGT
250
AATTGATGTG
CCAATACTTA
TCAGTGCGAT
CATACCAGCA
CTAAAa
Fig. 1. Nucleotide sequence of 5S rRNA gene repeat of Glycine soja. The noncoding strand is shown. The primers corresponding to 3' and 5' sequences of the coding region as used for PCR are underlined. A and T residues added by Taq-polymerase are shown in lower case.
-35-AGAAGGAATATAGT-A * * ** AAAGG , ACAC
***** GATATATGGA ****** CATATATTCG
ATTGATGTGC , **
**
CAATACTTAT-I *
ATCGAGAGTC ,
, ,
ACTGTGTTGC
****
ATGTACTAAC ,
G. S.
*
ize to the cloned NTS, but could be revealed by hybridization with the conserved coding region.
S. a .
, ******* TTCTACTAAC
M.i.
Fig. 2. Comparison of 5'-flanking regions of 5S rRNA genes of Glycine soja (G.s.), Sinapis alba (S.a.) [3] and Matthiola incana (M.i.) [4]. The 'TATA-Iike' sequence in G.s. is underlined. Asterisks mark identical nucleotides. Sequences are aligned according to existing data on the location of coding region. A deletion and an insertion were introduced in the G.s. sequence for better alignment.
Acknowledgements The authors thank the Racheff Endowment and the American Soybean Association for financial support. A.K. is on leave from Institute of General Genetics, Moscow, Russia.
References (Glycine m a x (L.) Merr. cv. Bragg) DNA with the
coding sequence from a distant plant (barley [5]), confirmed the length of the repeat obtained by sequencing (not shown). There were approximately 3 to 5 x 103 copies of 5S genes per haploid genome of both G. m a x and G. soja. Our results also showed that Hae III digestion produced different patterns with G. m a x and G. soja. These species are closely related, give fertile progeny, have been used for RFLP mapping of the soybean genome, and are considered to be subspecies [8]. Therefore, the polymorphism of 5S rRNA genes may be used for genetic mapping. Comparison of the hybridization pattern of G. m a x and G. soja DNA with the 5S coding region and NTS suggests that there exists another minor family of 5S repeats which did not hybrid-
1. Bogenhagen DF, Brown DD: Sequences in Xenopus 5S D N A required for transcription termination. Cell 24: 261-270 (1981). 2. Brown DD: The role of stable complexes that repress and activate eukaryotic genes. Cell 37:359-365 (1984). 3. Capesius I: Sequence of the 5S rRNA gene from Sinapsis alba. Plant Mol Biol 17:169-170 (1991). 4. Hemleben V, Werts D: Sequence organization and putative regulatory elements in the 5S rRNA genes of two higher plants ( Vigna radiata and Matthiola incana). Gene 62:165-169 (1988). 5. Kolchinsky AM, Kanazin VI, Yakovleva EYu, Gazumyan AK, Kole C, Ananiev EV: 5S-genes are located on the second chromosome of barley. Theor Appl Genetics 80:333-336 (1990). 6. Kolchinsky A, Kolesnikova M, Ananiev E: The portraying of plant genomes using PCR amplification of 5 S genes. Genome 34:1028-1031 (1991). 7. Selker U, Morzycka-Wroblenska E, Stevens JN, Metzen-
1047 berg RL: An upstream signal is required for in vitro transcription of Neurospora 5S genes. Mol Gen Genet 205: 189-192 (1986). 8. Singh ILl, Hymowitz T: The genomic relationship between Glycine max (L.) Merr. and Glycine soja (Sieb and
Zucc.) as revealed by pachytene chromosome analysis. Theor Appl Genet 76:705-711 (1988). 9. Specht T, Wolters J, Erdmann V: Compilation of 5S rRNA and 5S rRNA gene sequences. Nucl Acids Res 18 (suppl.) 2215-2230 (1991).
1045
Update section Sequence
Nucleotide sequence of the 5S rRNA gene from
Glycine soja
Alexander Kolchinsky and Peter M. Gresshoff Plant Molecular Genetics and Center for Legume Research, University of Tennessee, Knoxville, TN 37901-1071, USA Received 5 March 1992; accepted in revised form 23 March 1992
Key words: soybean (Glycine sp.), non-transcribed spacer, PCR
The non-transcribed spacer (NTS) of the 5S gene from Glycine soja (Sieb and Zucc., PI468.397) was amplified using the polymerase chain reaction (PCR) with primers universal for plant nuclear 5S genes described earlier [6] The primers are complementary to the 3' and 5' ends of highly conservative coding region and amplify divergent NTS sequences. The amplified fragment was cloned into the pCR1000 vector (Invitrogen) designed for direct cloning of PCR products. The vector has hanging 3'-thymidine residues complementary to adenine residues added to the 5' ends of PCR products by Taq-polymerase. Both strands of the cloned NTS fragment were sequenced using the TaqTrack dideoxy-kit (Promega). The resulting nucleotide sequence for the G. soja 5S rRNA gene is shown in Fig. 1. The length of the cloned PCR product was 255 bp, the primers are 20 bp long each, therefore the presumed length of the NTS was 215 bp, and the whole repeat was 335 bp long. These estimates correspond to the length of initial PCR products used for cloning and their oligomers (not shown). The role of the 5'-flanking region in the transcription of 5S rRNA gene by RNA-poly-
merase III is not quite clear. In contrast to earlier suggestions (see [2]), more recent data indicate that the promoter is not confined to the coding region and involves 5'-flanking sequences [7]. The analysis of the NTS of G. soja sequenced in this work, and its comparison to 5' regions from other plants [3, 4], showed clear homology to putative promoters (Fig. 2). They contained a consensus 'TATA' sequence 29 bp upstream from beginning of transcription, found to be required for in vitro transcription in some species [7]. The sequences surrounding the start of transcription in these cases were also similar. However, some 5S sequences (for example, the barley gene [5]) did not contain any obvious TATA-Iike sequences at proper positions (see compilation in [9]). The transcription termination signal for RNApolymerase III is thought to be a run of 4-5 thymidines surrounded on both sides with 2-3 GC pairs [1]. The sequenced 3'-downstream region of the G. soja 5S rRNA gene contained a typical run of 5 thymidines with CC and CG sequences in the close vicinity on both sides. An unusually long run of 17 thymidine residues followed this termination signal. Southern hybridization of G. soja and soybean
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X64631.
1046 tTGGGAAGTCC.TCGTGTTGCA.CCTCTTTTTA.CGTTTTTTTT.TTTTTTTTTC.
50
TTTTTGCCCT.TATTCTGAGT.ATTTTTCTTT.GAAGCGAAGT.AAAAGGTCGA.
i00
TAAGTTAACT.AATTTTTGTG.ATTGATCGGG.AGATAAATGT.ATCGTGGCCC.
150
GTGCTCGTTC.GTGTAGAAGG.TCGATCGAAA.GTCGCCGTCC.GGCAGCAGAA.
200
GGAATATAGT
250
AATTGATGTG
CCAATACTTA
TCAGTGCGAT
CATACCAGCA
CTAAAa
Fig. 1. Nucleotide sequence of 5S rRNA gene repeat of Glycine soja. The noncoding strand is shown. The primers corresponding to 3' and 5' sequences of the coding region as used for PCR are underlined. A and T residues added by Taq-polymerase are shown in lower case.
-35-AGAAGGAATATAGT-A * * ** AAAGG , ACAC
***** GATATATGGA ****** CATATATTCG
ATTGATGTGC , **
**
CAATACTTAT-I *
ATCGAGAGTC ,
, ,
ACTGTGTTGC
****
ATGTACTAAC ,
G. S.
*
ize to the cloned NTS, but could be revealed by hybridization with the conserved coding region.
S. a .
, ******* TTCTACTAAC
M.i.
Fig. 2. Comparison of 5'-flanking regions of 5S rRNA genes of Glycine soja (G.s.), Sinapis alba (S.a.) [3] and Matthiola incana (M.i.) [4]. The 'TATA-Iike' sequence in G.s. is underlined. Asterisks mark identical nucleotides. Sequences are aligned according to existing data on the location of coding region. A deletion and an insertion were introduced in the G.s. sequence for better alignment.
Acknowledgements The authors thank the Racheff Endowment and the American Soybean Association for financial support. A.K. is on leave from Institute of General Genetics, Moscow, Russia.
References (Glycine m a x (L.) Merr. cv. Bragg) DNA with the
coding sequence from a distant plant (barley [5]), confirmed the length of the repeat obtained by sequencing (not shown). There were approximately 3 to 5 x 103 copies of 5S genes per haploid genome of both G. m a x and G. soja. Our results also showed that Hae III digestion produced different patterns with G. m a x and G. soja. These species are closely related, give fertile progeny, have been used for RFLP mapping of the soybean genome, and are considered to be subspecies [8]. Therefore, the polymorphism of 5S rRNA genes may be used for genetic mapping. Comparison of the hybridization pattern of G. m a x and G. soja DNA with the 5S coding region and NTS suggests that there exists another minor family of 5S repeats which did not hybrid-
1. Bogenhagen DF, Brown DD: Sequences in Xenopus 5S D N A required for transcription termination. Cell 24: 261-270 (1981). 2. Brown DD: The role of stable complexes that repress and activate eukaryotic genes. Cell 37:359-365 (1984). 3. Capesius I: Sequence of the 5S rRNA gene from Sinapsis alba. Plant Mol Biol 17:169-170 (1991). 4. Hemleben V, Werts D: Sequence organization and putative regulatory elements in the 5S rRNA genes of two higher plants ( Vigna radiata and Matthiola incana). Gene 62:165-169 (1988). 5. Kolchinsky AM, Kanazin VI, Yakovleva EYu, Gazumyan AK, Kole C, Ananiev EV: 5S-genes are located on the second chromosome of barley. Theor Appl Genetics 80:333-336 (1990). 6. Kolchinsky A, Kolesnikova M, Ananiev E: The portraying of plant genomes using PCR amplification of 5 S genes. Genome 34:1028-1031 (1991). 7. Selker U, Morzycka-Wroblenska E, Stevens JN, Metzen-
1047 berg RL: An upstream signal is required for in vitro transcription of Neurospora 5S genes. Mol Gen Genet 205: 189-192 (1986). 8. Singh ILl, Hymowitz T: The genomic relationship between Glycine max (L.) Merr. and Glycine soja (Sieb and
Zucc.) as revealed by pachytene chromosome analysis. Theor Appl Genet 76:705-711 (1988). 9. Specht T, Wolters J, Erdmann V: Compilation of 5S rRNA and 5S rRNA gene sequences. Nucl Acids Res 18 (suppl.) 2215-2230 (1991).
