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A yeast model for classical juvenile Batten disease(CLN3)
doi: 10.1053/ejpn.2000.0448 available online at http://www.idealibrary.com on I l l ~ l " European Journal of Paediatric Neurology 2001; 5(Suppl. A): 127-129 ORIGINAL
ARTICLE
A yeast model for classical juvenile Batten disease (CLN3) KAREN J BARWELL, M U R R A Y
F BROOM
Biochemistry Department, University of Otago, Dunedin, New Zealand
The gene involved in the classical juvenile form of Batten disease, CLN3 has been identified as being highly homologous to the Saccharomyces cerevisiae YHC3 gene. To provide a simple model for the biochemical events underlying this disease, several disruptions have been made in YHC3 in three different S. cerevisiae strains. No obvious growth differences were observed, and neither was the previously reported phenotypic difference between wild-type and yeast disrupted in YHC3.
Keywords:Battendisease.Neuronalceroid lipofuscinosis.Saccharoraycescerevisiae.
Introduction Batten disease, or the neuronal ceroid lipofuscinoses (NCLs), are a group of severe degenerative neurological diseases affecting children and animals. There are eight genetically distinct forms of the disease, all of which are autosomal recessively inherited and characterized by progressive loss of vision, seizures, and psychomotor disturbances. The variants are distinguished by age of onset, and subtle differences in pathology and neurological symptoms. Neurologically the NCLs show striking degeneration of nerve cells and cortical atrophy, and at a biochemical level there is widespread accumulation of autofluorescent lysosomal lipopigments in neurons and many other cell types. In the majority of forms of the disease the dominant lysosomal storage material is subunit c, the hydrophobic intramembrane channel polypeptide of the mitochondrial ATP synthase. 1 The link between intracellular accumulation of subunit c and the neuropathology is unknown. The gene involved in the juvenile form of NCL (CLN3) has been cloned and identified as being highly homologous to YHC3, a gene of unknown function in Saccharomyces cerevisiae. 2 This conservation suggests that the CLN3 gene product has a
ubiquitous role throughout eukaryotes, and implies that knocking out the yeast YHC3 gene may provide a simple biochemical model for the cellular role of CLN3. Accordingly, we have disrupted the yeast YHC3 gene by transformation and homologous recombination in an effort to identify a phenotype that will indicate the role of YHC3 in the cell. The most common human mutation of CLN3 results in a 1.02-kb deletion. 3 A similar truncation has been created in the yeast YHC3, in addition to creating a yeast with the entire gene deleted. Previously reported growth differences between wild type and knockout yeast grown in the presence of D-(-)-threo-2-amino-l-[p-nitrophenyl]-l,3-propanediol (denoted ANP) 4 were not observed.
Materials and methods Yeast strains
Strains of S. cerevisiae used to construct the yeast model of Batten disease are as follows: AB1380: M A T a ~/+ ura3 trpl ade2-1 canl-lO0 lys2-1 his5, 5 GSY112: M A T ~ pep4::HIS3 prbl-A1.6R leu2::hisG
Correspondence:KJ Barwell, BiochemistryDepartment,Universityof Otago, PO Box56, Dunedin, New Zealand [email protected] 1090-3798/01/05/127+3 $35.00
© 2001 European Paediatric Neurology Society
128
ura3 can1 cir, °6 DBY746: MAT a his3-A1 leu2-3 Ieu2112 ura3-52 trp1-289. 7
YHC3 gene disruption Disruption of the yeast YHC3 gene was achieved by generating a linear transformation construct by polymerase chain reaction (PCR). s This construct contained the gene for the selectable marker URA3 from Candida albicans, flanked by 40 bp of YHC3 at each end. Forward and reverse primers for the KO construct were TTA GGA ATA TCT TTC GCA TCT ATA TCT TCC GGA T I T GGA GTT GCT GTA GTG CCA TTG and AGT TAG CTF CAC TGG AAC T I T AAA GAT TGA AGT TAA GAA AAG GAC CAC CTT TGA TTG respectively. Forward and reverse primers for the A construct were GT]? TTG AAC AGT GTA T I T ATA GAG TAC TTA AAC ACA TAT GTT GCT GTA GTG CCA TTG and TCG AAG AGT TAT TAG CAG GCA TFA GAA TGA GTA TAT AGA ACT TCA CAT TTA TAA TTG GC respectively. Forward and reverse primers for the H construct were TTA GGA ATA TCT TTC GCA TCT ATA TCT TCC GGA T I T GGA GT]? GCT GTA GTG CCA TTG and TCG AAG AGT TAT TAG CAG GCA TTA GAA TGA GTA TAT AGA ACT TCA CAT TTA TAA TTG GC respectively. The PCR conditions were 1 cycle of 4 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and I minute at 72°C. The PCR mix was directly used to transform yeast using a lithium acetate transformation procedure. 9
Genotype confirmation Genomic DNA was extracted from all yeast colonies TM and genotypes determined by PCR amplification of the YHC3 gene. The YHC3F primer was GGA ATT CAT TCC ATA TTT TCT TCG CGT A and the YHC3R primer was GGG TAC CTC GAA GAG TTA TTA GCA GGC AT. Conditions for PCR were 30 cycles of 45 seconds at 95°C, 1 minute at 65°C, and 2 minutes at 72°C.
Growth curves An overnight culture grown in YPD (1% Bacto Yeast Extract, 2% Bacto Peptone, and 2% glucose) was washed twice with sterile water after centrifugation for 5 min at 2000 rpm. The cells were resuspended in water at a titre of 5 x 107 cells/ml so that inoculation of 50 ml YPD could be achieved at a titre of I x 105 cells/ml. The 50 ml of inoculated YPD was grown in a sterile 250 ml flask, with shaking at 27°C. Growth was assessed by measuring A610
Original article: K J Barwell, Murray F Broom values on a LKB Biochrom Ultrospec II spectrophotometer.
Results YHC3 gene disruption Three transformation constructs, KO, H, and A were generated by PCR. Homologous recombination of the KO construct results in the deletion of 105 bp in the middle of the YHC3 gene and the insertion of URA3. This insertion would result in only the first 107 amino acids of the YHC3 gene product being translated, along with additional 19 novel amino acids before a premature stop codon is encountered. The most common hum an mutation of CLN3 results in a 1.02-kbp deletion and is found on at least one chromosome in 96% of CLN3 patients. 3 The predicted truncated gene product generated by this mutation contains only the first 153 amino acids, which are equivalent to the first 95 amino acids in the yeast. This KO construct can, therefore, be considered to create a similar mutation to the most common human mutation. The construct H results in all but the first 232 bp of the coding sequence of the YHC3 gene being deleted, and the insertion of URA3. The gene product, however, would be the same as that for the KO construct. The construct A removes the entire coding sequence of YHC3 and thus no gene product should be translated. Three different yeast strains were used to create various YHC3 mutations. The strains AB1380 and GSY112 were mutated using the KO construct, while DBY746 was mutated using the A and H constructs. Genotypes of colonies were confirmed by PCR amplification of YHC3 (data not shown).
Phenotypic examination Growth curves were carried out in YPD adjusted to pH 7.0. No obvious growth differences were seen. Growth differences between AYHC3 and wild-type yeast when grown in YPD in the presence of 0. 66m g/ m l ANP have been reported. 4 Figure 1 shows that this growth difference was not observed in any of the three yeast strains, or with any of the different constructs.
Discussion A YHC3 gene disruption has been created in yeast that results in a truncated gene product equivalent
Original article: A yeast model for classical juvenile Batten disease
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juvenile N C L (CLN3) has b e e n k n o w n since 19952 the role of the gene p r o d u c t is still not clear. The yeast m o d e l has p r o v i d e d s o m e insights into the biochemical role of CLN3 b a s e d on the o b s e r v e d A N P p h e n o t y p e in A Y H C 3 yeast, 11 and a l t h o u g h this has not b e e n r e p r o d u c e d in all strains, the yeast m o d e l is a simple s y s t e m a n d m a y still yield useful biochemical information a b o u t classical juvenile NCL.
Acknowledgements
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2 0 1
2
3
4
time ( d a y )
We a c k n o w l e d g e funding s u p p o r t f r o m the Wellc o m e Trust a n d the Batten Disease S u p p o r t a n d Research Association. KJB w a s a recipient of a University of Otago P o s t g r a d u a t e Scholarship.
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Fig. 1. Growth curves of wild-type (open diamond) and AYHC3 (filled symbols) yeast strains in liquid YPD (solid lines), and YPD containing 0.66 mg/ml ANP (dotted lines). A) Knockout construct KO was introduced to yeast strain AB1380. Three different knockout colonies (triangle, square, and circle) were compared with wild-type (open diamond). B) Knockout construct KO was introduced to yeast strain GSY112. Three different knockout colonies (triangle, square, and circle) were compared with wild-type (open diamond). C) Knockout construct A (triangle) and H (square) were introduced to yeast strain DBY746. Both were compared with wild-type (open diamond). No difference was observed in the growth of any knockout colony compared with the wild-type under the same conditions.
to the truncated gene p r o d u c t generated b y the m o s t c o m m o n h u m a n m u t a t i o n of CLN3. This has been r e p e a t e d in three different yeast strains. Furthermore, the entire Y H C 3 gene has b e e n deleted in one strain. Previously r e p o r t e d g r o w t h differences b e t w e e n w i l d - t y p e a n d k n o c k o u t yeast g r o w n in the presence of A N P 4 w a s not o b s e r v e d in a n y strain, or w i t h a n y of the gene deletions. This suggests the possibility that the A N P effect p r e v i o u s l y r e p o r t e d m a y be strain-specific. Despite the fact that the gene responsible for classical
References 1 Jolly RD. The ovine model of neuronal ceroid lipofuscinosis (NCL): its contribution to understanding the pathogenesis of Batten disease. Neuropediatrics 1997; 28: 60-62. 2 International Batten Disease Consortium. Isolation of a novel gene underlying Batten disease. CLN3. Cell 1995; 82: 949-947. 3 Munroe PB, Mitchison HM, O'Rawe AM et al. Spectrum of mutations in the Batten disease gene, CLN3. Am J Hum Genet 1997; 61: 310-316. 4 Pearce DA, Sherman F. A yeast model for the study of Batten disease. Proc Natl Acad Sci USA 1998; 95: 6915--6918. 5 Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Biotechnology 1992; 24: 172-178. 6 Wagenbach M, O'Rourke K, Vitez Let al. Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae. Biotechnology (NY) 1991; 9: 57--61. 7 Mikus MD, Petes TD. Recombination between genes located on nonhomologous chromosomes in Saccharomyces cerevisiae. Genetics 1982; 101: 369-404. 8 Baudin A, Ozier KO, Denouel A et al. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 1993; 21: 3329-3330. 9 Gietz RD, Woods RA. High efficiency transformation with lithium acetate. In: Johnston JR (ed) Molecular Genetics Of Yeast: A Practical Approach. Oxford and New York: IRL Press at Oxford University Press, 1994: 121-134. 10 Wach A, Pick H, Philippsen P. In: Johnston JR (ed) Molecular Genetics of Yeast: A Practical Approach. Oxford and New York: IRL Press at Oxford University Press, 1994. 11 Pearce DA, Ferea T, Nosel SA et al. Action of BTN1, the yeast orthologue of the gene mutated in Batten disease. Nature Genet 1999; 22: 55.
ARTICLE
A yeast model for classical juvenile Batten disease (CLN3) KAREN J BARWELL, M U R R A Y
F BROOM
Biochemistry Department, University of Otago, Dunedin, New Zealand
The gene involved in the classical juvenile form of Batten disease, CLN3 has been identified as being highly homologous to the Saccharomyces cerevisiae YHC3 gene. To provide a simple model for the biochemical events underlying this disease, several disruptions have been made in YHC3 in three different S. cerevisiae strains. No obvious growth differences were observed, and neither was the previously reported phenotypic difference between wild-type and yeast disrupted in YHC3.
Keywords:Battendisease.Neuronalceroid lipofuscinosis.Saccharoraycescerevisiae.
Introduction Batten disease, or the neuronal ceroid lipofuscinoses (NCLs), are a group of severe degenerative neurological diseases affecting children and animals. There are eight genetically distinct forms of the disease, all of which are autosomal recessively inherited and characterized by progressive loss of vision, seizures, and psychomotor disturbances. The variants are distinguished by age of onset, and subtle differences in pathology and neurological symptoms. Neurologically the NCLs show striking degeneration of nerve cells and cortical atrophy, and at a biochemical level there is widespread accumulation of autofluorescent lysosomal lipopigments in neurons and many other cell types. In the majority of forms of the disease the dominant lysosomal storage material is subunit c, the hydrophobic intramembrane channel polypeptide of the mitochondrial ATP synthase. 1 The link between intracellular accumulation of subunit c and the neuropathology is unknown. The gene involved in the juvenile form of NCL (CLN3) has been cloned and identified as being highly homologous to YHC3, a gene of unknown function in Saccharomyces cerevisiae. 2 This conservation suggests that the CLN3 gene product has a
ubiquitous role throughout eukaryotes, and implies that knocking out the yeast YHC3 gene may provide a simple biochemical model for the cellular role of CLN3. Accordingly, we have disrupted the yeast YHC3 gene by transformation and homologous recombination in an effort to identify a phenotype that will indicate the role of YHC3 in the cell. The most common human mutation of CLN3 results in a 1.02-kb deletion. 3 A similar truncation has been created in the yeast YHC3, in addition to creating a yeast with the entire gene deleted. Previously reported growth differences between wild type and knockout yeast grown in the presence of D-(-)-threo-2-amino-l-[p-nitrophenyl]-l,3-propanediol (denoted ANP) 4 were not observed.
Materials and methods Yeast strains
Strains of S. cerevisiae used to construct the yeast model of Batten disease are as follows: AB1380: M A T a ~/+ ura3 trpl ade2-1 canl-lO0 lys2-1 his5, 5 GSY112: M A T ~ pep4::HIS3 prbl-A1.6R leu2::hisG
Correspondence:KJ Barwell, BiochemistryDepartment,Universityof Otago, PO Box56, Dunedin, New Zealand [email protected] 1090-3798/01/05/127+3 $35.00
© 2001 European Paediatric Neurology Society
128
ura3 can1 cir, °6 DBY746: MAT a his3-A1 leu2-3 Ieu2112 ura3-52 trp1-289. 7
YHC3 gene disruption Disruption of the yeast YHC3 gene was achieved by generating a linear transformation construct by polymerase chain reaction (PCR). s This construct contained the gene for the selectable marker URA3 from Candida albicans, flanked by 40 bp of YHC3 at each end. Forward and reverse primers for the KO construct were TTA GGA ATA TCT TTC GCA TCT ATA TCT TCC GGA T I T GGA GTT GCT GTA GTG CCA TTG and AGT TAG CTF CAC TGG AAC T I T AAA GAT TGA AGT TAA GAA AAG GAC CAC CTT TGA TTG respectively. Forward and reverse primers for the A construct were GT]? TTG AAC AGT GTA T I T ATA GAG TAC TTA AAC ACA TAT GTT GCT GTA GTG CCA TTG and TCG AAG AGT TAT TAG CAG GCA TFA GAA TGA GTA TAT AGA ACT TCA CAT TTA TAA TTG GC respectively. Forward and reverse primers for the H construct were TTA GGA ATA TCT TTC GCA TCT ATA TCT TCC GGA T I T GGA GT]? GCT GTA GTG CCA TTG and TCG AAG AGT TAT TAG CAG GCA TTA GAA TGA GTA TAT AGA ACT TCA CAT TTA TAA TTG GC respectively. The PCR conditions were 1 cycle of 4 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and I minute at 72°C. The PCR mix was directly used to transform yeast using a lithium acetate transformation procedure. 9
Genotype confirmation Genomic DNA was extracted from all yeast colonies TM and genotypes determined by PCR amplification of the YHC3 gene. The YHC3F primer was GGA ATT CAT TCC ATA TTT TCT TCG CGT A and the YHC3R primer was GGG TAC CTC GAA GAG TTA TTA GCA GGC AT. Conditions for PCR were 30 cycles of 45 seconds at 95°C, 1 minute at 65°C, and 2 minutes at 72°C.
Growth curves An overnight culture grown in YPD (1% Bacto Yeast Extract, 2% Bacto Peptone, and 2% glucose) was washed twice with sterile water after centrifugation for 5 min at 2000 rpm. The cells were resuspended in water at a titre of 5 x 107 cells/ml so that inoculation of 50 ml YPD could be achieved at a titre of I x 105 cells/ml. The 50 ml of inoculated YPD was grown in a sterile 250 ml flask, with shaking at 27°C. Growth was assessed by measuring A610
Original article: K J Barwell, Murray F Broom values on a LKB Biochrom Ultrospec II spectrophotometer.
Results YHC3 gene disruption Three transformation constructs, KO, H, and A were generated by PCR. Homologous recombination of the KO construct results in the deletion of 105 bp in the middle of the YHC3 gene and the insertion of URA3. This insertion would result in only the first 107 amino acids of the YHC3 gene product being translated, along with additional 19 novel amino acids before a premature stop codon is encountered. The most common hum an mutation of CLN3 results in a 1.02-kbp deletion and is found on at least one chromosome in 96% of CLN3 patients. 3 The predicted truncated gene product generated by this mutation contains only the first 153 amino acids, which are equivalent to the first 95 amino acids in the yeast. This KO construct can, therefore, be considered to create a similar mutation to the most common human mutation. The construct H results in all but the first 232 bp of the coding sequence of the YHC3 gene being deleted, and the insertion of URA3. The gene product, however, would be the same as that for the KO construct. The construct A removes the entire coding sequence of YHC3 and thus no gene product should be translated. Three different yeast strains were used to create various YHC3 mutations. The strains AB1380 and GSY112 were mutated using the KO construct, while DBY746 was mutated using the A and H constructs. Genotypes of colonies were confirmed by PCR amplification of YHC3 (data not shown).
Phenotypic examination Growth curves were carried out in YPD adjusted to pH 7.0. No obvious growth differences were seen. Growth differences between AYHC3 and wild-type yeast when grown in YPD in the presence of 0. 66m g/ m l ANP have been reported. 4 Figure 1 shows that this growth difference was not observed in any of the three yeast strains, or with any of the different constructs.
Discussion A YHC3 gene disruption has been created in yeast that results in a truncated gene product equivalent
Original article: A yeast model for classical juvenile Batten disease
6
4
;
_
2
~-;~:;t
-
_
:
=
=
_-
: :~il~: : " ~ : 1 1 . ~ " " - I I - - i
0 1
2 time (day)
3
4
B lo
1 2 9
juvenile N C L (CLN3) has b e e n k n o w n since 19952 the role of the gene p r o d u c t is still not clear. The yeast m o d e l has p r o v i d e d s o m e insights into the biochemical role of CLN3 b a s e d on the o b s e r v e d A N P p h e n o t y p e in A Y H C 3 yeast, 11 and a l t h o u g h this has not b e e n r e p r o d u c e d in all strains, the yeast m o d e l is a simple s y s t e m a n d m a y still yield useful biochemical information a b o u t classical juvenile NCL.
Acknowledgements
6 O
2 0 1
2
3
4
time ( d a y )
We a c k n o w l e d g e funding s u p p o r t f r o m the Wellc o m e Trust a n d the Batten Disease S u p p o r t a n d Research Association. KJB w a s a recipient of a University of Otago P o s t g r a d u a t e Scholarship.
C 12
10
-
~
-
~6
2 6
-
,
-
1 time
,
,
2
3
(day)
Fig. 1. Growth curves of wild-type (open diamond) and AYHC3 (filled symbols) yeast strains in liquid YPD (solid lines), and YPD containing 0.66 mg/ml ANP (dotted lines). A) Knockout construct KO was introduced to yeast strain AB1380. Three different knockout colonies (triangle, square, and circle) were compared with wild-type (open diamond). B) Knockout construct KO was introduced to yeast strain GSY112. Three different knockout colonies (triangle, square, and circle) were compared with wild-type (open diamond). C) Knockout construct A (triangle) and H (square) were introduced to yeast strain DBY746. Both were compared with wild-type (open diamond). No difference was observed in the growth of any knockout colony compared with the wild-type under the same conditions.
to the truncated gene p r o d u c t generated b y the m o s t c o m m o n h u m a n m u t a t i o n of CLN3. This has been r e p e a t e d in three different yeast strains. Furthermore, the entire Y H C 3 gene has b e e n deleted in one strain. Previously r e p o r t e d g r o w t h differences b e t w e e n w i l d - t y p e a n d k n o c k o u t yeast g r o w n in the presence of A N P 4 w a s not o b s e r v e d in a n y strain, or w i t h a n y of the gene deletions. This suggests the possibility that the A N P effect p r e v i o u s l y r e p o r t e d m a y be strain-specific. Despite the fact that the gene responsible for classical
References 1 Jolly RD. The ovine model of neuronal ceroid lipofuscinosis (NCL): its contribution to understanding the pathogenesis of Batten disease. Neuropediatrics 1997; 28: 60-62. 2 International Batten Disease Consortium. Isolation of a novel gene underlying Batten disease. CLN3. Cell 1995; 82: 949-947. 3 Munroe PB, Mitchison HM, O'Rawe AM et al. Spectrum of mutations in the Batten disease gene, CLN3. Am J Hum Genet 1997; 61: 310-316. 4 Pearce DA, Sherman F. A yeast model for the study of Batten disease. Proc Natl Acad Sci USA 1998; 95: 6915--6918. 5 Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Biotechnology 1992; 24: 172-178. 6 Wagenbach M, O'Rourke K, Vitez Let al. Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae. Biotechnology (NY) 1991; 9: 57--61. 7 Mikus MD, Petes TD. Recombination between genes located on nonhomologous chromosomes in Saccharomyces cerevisiae. Genetics 1982; 101: 369-404. 8 Baudin A, Ozier KO, Denouel A et al. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 1993; 21: 3329-3330. 9 Gietz RD, Woods RA. High efficiency transformation with lithium acetate. In: Johnston JR (ed) Molecular Genetics Of Yeast: A Practical Approach. Oxford and New York: IRL Press at Oxford University Press, 1994: 121-134. 10 Wach A, Pick H, Philippsen P. In: Johnston JR (ed) Molecular Genetics of Yeast: A Practical Approach. Oxford and New York: IRL Press at Oxford University Press, 1994. 11 Pearce DA, Ferea T, Nosel SA et al. Action of BTN1, the yeast orthologue of the gene mutated in Batten disease. Nature Genet 1999; 22: 55.