4. Material & Methods
4.1 Material
4.1.4 Oligonucleotides
4.1.4.1 Primer for PCR amplification and sequencing
# Name 5’-3’-sequence Purpose
M13-21/M13 TGTAAAACGACGGCCAG forward primer provided for sequencing by Thermo Fisher GENEART and Microsynth SEQLAB
M13-R/M13r CAGGAAACAGCTATGAC reverse primer provided for sequencing by Thermo Fisher GENEART and Microsynth SEQLAB
#621 Noc2-TAP FP AAGTGATGATGACAACGAAGATG TTGAAATGTCAGACGCTTCCATGG AAAAGAGAAG
forward primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of NOC2
# Name 5’-3’-sequence Purpose #622 Noc2-TAP RP CTATTGAATTCAAGACAAAAAATC
AAATCTTGCTGAGTTGTACGACTC ACTATAGGG
reverse primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of NOC2
#1203 RPA135-pYM-for CTATCCGCAATGGGTATAAGATTG CGTTATAATGTAGAGCCCAAACGT ACGCTGCAGGTCGAC
forward primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of RPA135
#1204 RPA135-pYM-rev CCTTCATTTACCATTCTATATCAA TTTGGAAAGAAGGGTATTTCTATC GATGAATTCGAGCTC
reverse primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of RPA135
#1813 Utp4-TAP-F ACTTTTCACTCCAAACAAAAGGCG TTTATTCAACCAAAGTTAGTGTTT TCCATGGAAAAGAGAAG
forward primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of UTP4
#1814 Utp4-TAP-R GCCTTTTAATAGCATCTCTCTATT CTTCGGTATGTTGACTTAAATTAA TACGACTCACTATAGGG
reverse primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of UTP4
#2934 NOP53-TAP_fw AGTGCCCGTTAGGAAAGGTAGAA AGTATAAGCAGAAAATCACTGAA AAGTGGACACATAAGGACTTCAA ATCCATGGAAAAGAGAAG
forward primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of NOP53
#2935 NOP53-TAP_rev ATCTCACTTGATGAATCCACGTAT CAAGGACAACCTTTTCATGGAAAC ATATACTGTAAAACAAAAAACTT ACGACTCACTATAGGG
reverse primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of NOP53
#3696 UTP5-S3-fw AGCGACGGCGAGGAGGAAGCCGGA TATAGTGACGTTGAGATGGAACG TACGCTGCAGGTCGAC
forward primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP5
#3697 UTP5-S2-rev ATTTTTGTATTCTGATGCGTGAAA GCATTTTATGCATGATATCCTATC GATGAATTCGAGCTCG
reverse primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP5
#3698 UTP8-S3-fw CAGAAGCGAGCCTTACCCACTTAC ACCATGGAATACTTGGACATTCGT ACGCTGCAGGTCGAC
forward primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP8
#3699 UTP8-S2-rev TTTTCTATATAGGTATTATACAAT ACAATCAAATTCATTGCATACATC GATGAATTCGAGCTCG
reverse primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP8
#3702 UTP10-S3-fw GTTGTTGAAAACGTTTTAGGGGA ACCTTTTGATAGGTATTTAGATCG TACGCTGCAGGTCGAC
forward primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP10
#3703 UTP10-S2-rev GTGTTTTACTTTACAAAAATTTAC ATAATACTTCACCTTTTTTTTATC GATGAATTCGAGCTCG
reverse primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of UTP10
#3883 S3_TAP CAGCTGAAGCTTCGTACGCTGCAG GTCGACATGGAAAAGAGAAGATG GAAAAAG
forward primer used to amplify TAP tag from pBS1539
#3884 S2_TAP GGATCTGATATCATCGATGAATTC GAGCTCGATACGACTCACTATAGG GCG
reverse primer used to amplify TAP tag from pBS1539
# Name 5’-3’-sequence Purpose
#3890 Pol5(2529)_fo CGTTATCTCGTTCAAGCATAGAG forward primer for sequencing of mutated pol5 on pCMS16 and ptCMS2
#3891 Pol5(3380)_re ACAAACACCTGATGACGCGG reverse primer for sequencing of mutated pol5 on pCMS16 and ptCMS2
#3912 Pol5(572)_fo TATGGATGAAAGTTCCGCTG forward primer for sequencing of mutated pol5 on pCMS16 and ptCMS2
#3913 Pol5(2634)_re ACGAGCTTGCTTAATGAGCC reverse primer for sequencing of mutated pol5 on pCMS16 and ptCMS2
#4072 RLP7_For_Int CCAGTTATCGAAGTTGACATTGAC TCTTTATTAGCCAAGTTGAATTCC ATGGAAAAGAGAAG
forward primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of RLP7
#4073 RLP7_Rev_Int TAACTAACAACCTATGTACTATAC AATTTTAAAATACTCTCTTAATAC GACTCACTATAGGG
reverse primer used to amplify TAP tag from pBS1539 for yeast homologous recombination at the C-terminus of RLP7
o86 FLAG_POL5_F ATGGATTACAAGGATGACGACGA TAAGGGTACCGGATCCATGACAGG GAAAGTCAACAGAG
forward primer used to amplify
POL5 from genomic DNA of
BY4741 for cloning into pCM182- LEU2
o87 NotI_POL5_Re GGAAACAGCTATGACCATGATTAC GCCAAGCTTGCATGCGCGGCCGCA GGTATGGACTCGTATGTTTATC
reverse primer used to amplify
POL5 from genomic DNA of
BY4741 for cloning into pCM182- LEU2
o99 Pol5_713_rev TTCAAATCCAACACCGAGGG reverse primer for sequencing of mutated pol5 on pCMS16 and ptCMS2
o127 Spb4_S3_fw CGGAAGAAAGTTTCCAGCAAAGCT ATCCAAGGCAATTTTGACGACTTA CGTACGCTGCAGGTCGAC
forward primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of SPB4
o128 Spb4_S2_rev CCATTGGTTAAGAATGTTGAGTGA TTCTACGAACAAGGTAACTTTTTT TCCATAAATCGATGAATTCGAGCT CG
reverse primer used to amplify TAP tag from pYM15-TAP-URA for yeast homologous recombination at the C-terminus of SPB4
o131 Pol5_DCt_PmeI_fw GTTTAAACCTAAGTATATTTTATC
ACTCCTCAAAATCGG forward primer used to amplify POL5 from pCMS16 for C-terminal
truncation of POL5
o132 Pol5_DCt120_rev GAGGAGTGATAAAATATACTTAG GTTTAAACTGGCCTATCAAGAGTT TCATTGAC
reverse primer used to amplify
POL5 from pCMS16 for C-terminal
truncation of last 120 amino acids of POL5
o133 Pol5_DCt252_rev GAGGAGTGATAAAATATACTTAG GTTTAAACGGGTAAATTTAACGCC TTCACTAAGGC
reverse primer used to amplify
POL5 from pCMS16 for C-terminal
truncation of last 252 amino acids of POL5
o134 Pol5_DCt343_rev GAGGAGTGATAAAATATACTTAG GTTTAAACCAAACCAACTTCTTCA ATAAACTGC
reverse primer used to amplify
POL5 from pCMS16 for C-terminal
truncation of last 343 amino acids of POL5
o135 Pol5_DNt137_fw CCTGCAGCCCGGGGGATCCATGCC TTTATTCTCAGAAATATTTGTTAA AGATTTGG
forward primer used to amplify
POL5 from pCMS16 for N-terminal
truncation of first 137 amino acids of POL5
# Name 5’-3’-sequence Purpose o136 Pol5_DNt293_fw CCTGCAGCCCGGGGGATCCATGCT
GCTACCGTTGTTTGGTAATGG forward primer used to amplify POL5 from pCMS16 for N-terminal
truncation of first 293 amino acids of POL5
o137 Pol5_DNt658_fw CCTGCAGCCCGGGGGATCCATGAA
GGCCTTACTCAGGAAATTGAG forward primer used to amplify POL5 from pCMS16 for N-terminal
truncation of first 658 amino acids of POL5
o138 Pol5_DNt_rev CATGGATCCCCCGGGCTGCAGGAA
TTCG reverse primer used to amplify POL5 from pCMS16 for N-terminal
truncation of POL5
o152 Seq_ptets CAAAGTCGAGTTTCTCGATCG forward primer for sequencing of plasmids containing tet-promoter
o249 Pol5_DN38_rev GAGGAGTGATAAAATATACTTAG GTTTAAACGTGTTTGGAAAATTCG CAAAGTTC
reverse primer used to amplify
POL5 from pCMS16 for C-terminal
truncation of pol5-D679L
o250 Pol5_DN75_rev GAGGAGTGATAAAATATACTTAG GTTTAAACAGTTCTTGATCTGCAA ACTTTACT
reverse primer used to amplify
POL5 from pCMS16 for C-terminal
truncation of pol5-D679L
o251 Pol5_DC50_fw CCTGCAGCCCGGGGGATCCATGAG TGAAAGTGAAAGTGAGAGTGATA GCGATGAT
forward primer used to amplify
POL5 from pCMS16 for N-terminal
truncation of pol5-DN677
o252 Pol5_DC92_fw CCTGCAGCCCGGGGGATCCATGCC CGATAACATAGTTAATGATAAAG GGGAAGTT
forward primer used to amplify
POL5 from pCMS16 for N-terminal
truncation of pol5-DN677
Table 3: Primer for PCR amplification and sequencing used in this study.
Oligonucleotides marked with “#” belong to the department of Biochemistry III at the University of Regensburg. Oligonucleotides marked with “o” belong to the collection of the UTP lab.
4.1.4.2 Primer for qPCR amplification
# Name 5’-3’-sequence Purpose #710
(“25S”) M1 TGGAGCAAAGAAATCACCGC reverse primer for amplification within 25S rDNA locus; position +6414 from ATG
#711
(“25S”) M2 CCGCTGGATTATGGCTGAAC forward primer for amplification within 25S rDNA locus; position +6366 from ATG
#712
(“18S”) M3 GAGTCCTTGTGGCTCTTGGC forward primer for amplification within 18S rDNA locus; position +1431 from ATG
#713
(“18S”) M4 AATACTGATGCCCCCGACC reverse primer for amplification within 18S rDNA locus; position +1589 from ATG
#920
(“5S”) 5S ChIP-F1 GCCATATCTACCAGAAAGCACC forward primer for amplification within 5S rDNA locus; position -1357 from ATG
#921
(“5S”) 5S ChIP-R1 GATTGCAGCACCTGAGTTTCG reverse primer for amplification within 5S rDNA locus; position -1265 from ATG
#969 (“Pro- moter”)
Prom ChIP-F2 TCATGGAGTACAAGTGTGAGGA forward primer for amplification within rDNA promoter; position -43 from ATG
#970 (“Pro- moter”)
Prom ChIP-R1 TAACGAACGACAAGCCTACTC reverse primer for amplification within rDNA promoter; position +70 from ATG
# Name 5’-3’-sequence Purpose #1049
(“ITS1”) R25 GCTTAGAGAAGGGGGCAACT forward primer for amplification at the end of 18S rDNA locus; position +2404 from ATG
#2421
(“E-Pro”) E-Pro_2_fwd CAGAGAGGCAGCGTAAAAGG forward primer for amplification within E-Pro downstream of rDNA terminator; +7245 from ATG
#2422
(“E-Pro”) E-Pro_2_rev CTTATTCCTTCCCGCTTTCC reverse primer for amplification within E-Pro downstream of rDNA terminator; +7457 from ATG
#2863
(“ITS1”) R25-rev-q CGGCTGGACTCTCCATCTCT reverse primer for amplification at the end of 18S rDNA locus; position +2596 from ATG
#2864
(“ITS2”) ITS2-up-q GCATGCCTGTTTGAGCGTC forward primer for amplification at the end of 5.8S locus; position +3015 from ATG
#2865
(“ITS2”) ITS2-do-q CGACCGTACTTGCATTATACC reverse primer for amplification at the end of 5.8S locus; position +3153 from ATG
#2882
(“3’ETS”) 25S-Jo-up ATCATTTGTATACGACTTAGATGTACAACG forward primer for amplification at the end of 25S rDNA locus; position +6576 from ATG
#2883
(“3’ETS”) 25S-Jo-do AACAAATCAGACAACAAAGGCTTAATC reverse primer for amplification at the end of 25S rDNA locus; position +6648 from ATG
#2884 (“Termi- nator”)
3’-REB-Jo-up TACGATGAGGATGATAGTGTGTAAG
AGTG forward primer for amplification within rDNA terminator; position +6821 from ATG
#2885 (“Termi- nator”)
3’-REB-Jo-do TCTCTTTCAACCCATCTTTGCAA reverse primer for amplification within rDNA terminator; position +6917 from ATG
#2886
(“NTS1”) Fob-bs-Jo-up GAGAAAAGCTCATTTCCTATAGTTAACAG forward primer for amplification within NTS1 downstream of rDNA terminator; position +7066 from ATG
#2887
(“NTS1”) Fob-bs-Jo-do TTCACTTGTCTCTTACATCTTTCTTGG reverse primer for amplification within NTS1 downstream of rDNA terminator; position +7138 from ATG
#3249
(“NTS2”) rARS1_fwd TGACGGAAATACGCTTCAGA reverse primer for amplification within NTS2 upstream of rDNA promoter; position -473 from ATG
#3250
(“NTS2”) rARS1_rev GTCAGATGAAAGATGAATAGACA forward primer for amplification within NTS2 upstream of rDNA promoter; position -609 from ATG
Table 4: Primer for qPCR amplification used in this study.
Oligonucleotides marked with “#” belong to the department of Biochemistry III at the University of Regensburg. Names in brackets correspond to denomination in qPCR diagrams.
4.1.4.3 Probes for northern blot detection and primer extension
# Name 5’-3’-sequence Hybridization site #205
(“18S”) o2-18S CATGGCTTAATCTTTGAGAC rDNA, 18S #207
# Name 5’-3’-sequence Hybridization site #208
(“A3B”) o5-A3/B1 AATTTCCAGTTACGAAAATTCTTG rDNA, ITS1 #210
(“EC2”) o7-E/C2 GGCCAGCAATTTCAAGTTA rDNA, ITS2 #211
(“C2C1”) o8-C1/C2 GAACATTGTTCGCCTAGA rDNA, ITS2 #212
(“25S”) o9-25S CTCCGCTTATTGATATGC rDNA, 25S #2474
(“5S”) 5S-rDNA CAGCGGGTACTCCTACCTGATT rDNA, 5S #2921
(“A0A1”) A0A1-Sonde CCCACCTATTCCCTCTTGCTAG rDNA, 5’ETS #2959
(“5.8S”) 5.8S rRNA 3’ AAATGACGCTCAAACAGGCAT rDNA, 5.8S #3468
(“U3”) U3-SnR17A CCGCTAAGGATTGCGGACCAAGC U3 #3470
(“U14”) SnR128-U14 TCACTCAGACATCCTAGGAAGG U14 #3839
(“DA2”) ITS1 D-A2 mid AAGCCTAGCAAGACCGCGCA rDNA, ITS1 o67
(“bA0”) vA0 CGCTGCTCACCAATGG rDNA, 5‘ETS Table 5: Probes for northern blot detection and primer extension used in this study.
Oligonucleotides marked with “#” belong to the department of Biochemistry III at the University of Regensburg. Oligonucleotides marked with “o” belong to the collection of the UTP lab.