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Expression profile of Caenorhabditis elegans mutant for the Werner syndrome gene ortholog reveals the impact of vitamin C on development to increase life span.

Dallaire A, Proulx S, Simard MJ, Lebel M - BMC Genomics (2014)

Bottom Line: We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals.Proteolysis was the only biological process significantly affected by vitamin C in wild type worms.Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche sur le Cancer de l'Université Laval, Hôpital Hôtel-Dieu de Québec (CHU de Québec Research Center), 9 McMahon Sreet, Québec City G1R 2 J6, Canada. michel.lebel@crhdq.ulaval.ca.

ABSTRACT

Background: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase ortholog also exhibits a shorter life span, which can be rescued by vitamin C. In this study, we analyzed the impact of a mutation in the wrn-1 gene and the dietary supplementation of vitamin C on the global mRNA expression of the whole C. elegans by the RNA-seq technology.

Results: Vitamin C increased the mean life span of the wrn-1(gk99) mutant and the N2 wild type strains at 25°C. However, the alteration of gene expression by vitamin C is different between wrn-1(gk99) and wild type strains. We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals. Such genes significantly affected the metabolism of lipid, cellular ketone, organic acid, and carboxylic acids. Vitamin C, in return, altered the expression of genes in wrn-1(gk99) worms involved in locomotion and anatomical structure development. Proteolysis was the only biological process significantly affected by vitamin C in wild type worms.

Conclusions: Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms. Finally, vitamin C extended the life span of wrn-1(gk99) animals by altering biological processes involved mainly in locomotion and anatomical structure development.

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Related in: MedlinePlus

Examples of RT-PCR products on 2% agarose gel. Results are shown for nlp-32, pes-8, cyp34a9, cyp-33e2, sodh-1, acs-2, ugt-33, acs-14, nas-3, and arrd-24 genes after 40 cycles of PCR. M = marker in base pairs; N2 = wild type strain; wrn-1 = wrn-1(gk99) C. elegans strain.
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Fig2: Examples of RT-PCR products on 2% agarose gel. Results are shown for nlp-32, pes-8, cyp34a9, cyp-33e2, sodh-1, acs-2, ugt-33, acs-14, nas-3, and arrd-24 genes after 40 cycles of PCR. M = marker in base pairs; N2 = wild type strain; wrn-1 = wrn-1(gk99) C. elegans strain.

Mentions: To gain insight into the rescuing effect of vitamin C on the life span of wrn-1(gk99) worms, we first established the global expression profile of untreated wild type and mutant worms at the L4 stage by RNA-seq. By carefully monitored physiological developmental cues (such as vulva and gonad development), we noticed a three hours developmental delay in wrn-1(gk99) mutants compared to wild type animals. Therefore, mRNA was extracted based on the biological age (i.e. the exact developmental stage) and not on the chronological age of the animals for the RNA-seq analyses. Table 1 contains the number of raw reads for each biological replicate (including the vitamin C-treated worms). The normalized counts for each biological replicate are shown in the Additional file1: Table S1 for all the C. elegans genes. A preliminary list of differentially expressed genes was generated by keeping the genes that showed a two-fold difference or more between wild type N2 and wrn-1(gk99) worms with an adjusted P-value <0.01. This first list contained 2528 genes (data not shown). We randomly chose 16 genes representing a range of base mean number from 4 to 3500 reads in both or in either N2 or wrn-1 mutant animals based on the RNA-seq normalized data (list of genes in Table 2) to determine the minimum of reads required for a gene to be detected by RT-PCR in our analyses. We thus designed 16 pairs of primers to validate the differential expression of these genes by quantitative RT-PCR. The tubulin encoding gene (tba-1) was included as a control (no difference in tba-1 expression between both strains). PolyA + RNA was thus extracted from three plates of wild type and from three plates of wrn-1(gk99) worms (each plate representing one biological replicate of worms at the L4 stage). The RT-PCR results are shown in Table 2 and the sequences of the primers are provided in the Additional file2: Table S2. Every RT-PCR product was examined on a 2% agarose gel at the end of each run. Figure 2 shows examples of such gels for ten of these genes. A PCR product was detected only in samples that showed more than 145 reads by RNA-seq (Additional file1: Table S1). For example, no band corresponding to nlp-32 mRNA could be detected with the mRNA samples and no Ct could be detected by quantitative RT-PCR. The mean number of reads is below 134 for nlp-32 mRNA in both the wild type and the wrn-1(gk99) worms (Table 2). Similarly, no arrd-24 mRNA could be detected from wild type or wrn-1(gk99) worms mRNA pools even after 40 cycles of PCR (Figure 2). The normalized counts for the arrd-24 gene were below 32 in both genotypes for all biological replicates (Table 2). No Ct could be detected by quantitative RT-PCR. In contrast, a band for the ugt-33 gene was detected with mRNA from the wild type worms only (no band was detected for the wrn-1(gk99) worms) (Figure 2). The mean number of reads was lower than 145 for ugt-33 in the wrn-1(gk99) worms but higher than 145 in the wild type worms (Table 2). Finally, Table 2 indicated expression differences for pes-8, cyp-34a9, cyp-33e2, sodh-1, ugt-33, ech-9, nas-3, gpx-3, acs-2, acs-14, asah-1, lys-8, Y46G5.20, and daf-9 genes between the wild type and the wrn-1(gk99) strains by RT-PCR analyses and such results confirmed the RNA-seq data. A mean number of reads greater than 145 was found in at least one of the genotypes following the RNA-seq analysis for these genes (Table 2). Based on such results, we generated a final list of genes showing at least a two-fold difference in expression between wild type and mutant worms, an adjusted P-value <0.01, and a mean number of reads >145 in at least one of the worm strain. These criteria were applied to each comparison hereafter in this report. Additional file3: Table S3 gives a final list of 1522 genes differentially expressed between wild type and wrn-1(gk99) worms. There were 907 down regulated and 615 up regulated genes respectively (by at least two-fold) in the wrn-1(gk99) worms compared to the wild type worms.Table 1


Expression profile of Caenorhabditis elegans mutant for the Werner syndrome gene ortholog reveals the impact of vitamin C on development to increase life span.

Dallaire A, Proulx S, Simard MJ, Lebel M - BMC Genomics (2014)

Examples of RT-PCR products on 2% agarose gel. Results are shown for nlp-32, pes-8, cyp34a9, cyp-33e2, sodh-1, acs-2, ugt-33, acs-14, nas-3, and arrd-24 genes after 40 cycles of PCR. M = marker in base pairs; N2 = wild type strain; wrn-1 = wrn-1(gk99) C. elegans strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Examples of RT-PCR products on 2% agarose gel. Results are shown for nlp-32, pes-8, cyp34a9, cyp-33e2, sodh-1, acs-2, ugt-33, acs-14, nas-3, and arrd-24 genes after 40 cycles of PCR. M = marker in base pairs; N2 = wild type strain; wrn-1 = wrn-1(gk99) C. elegans strain.
Mentions: To gain insight into the rescuing effect of vitamin C on the life span of wrn-1(gk99) worms, we first established the global expression profile of untreated wild type and mutant worms at the L4 stage by RNA-seq. By carefully monitored physiological developmental cues (such as vulva and gonad development), we noticed a three hours developmental delay in wrn-1(gk99) mutants compared to wild type animals. Therefore, mRNA was extracted based on the biological age (i.e. the exact developmental stage) and not on the chronological age of the animals for the RNA-seq analyses. Table 1 contains the number of raw reads for each biological replicate (including the vitamin C-treated worms). The normalized counts for each biological replicate are shown in the Additional file1: Table S1 for all the C. elegans genes. A preliminary list of differentially expressed genes was generated by keeping the genes that showed a two-fold difference or more between wild type N2 and wrn-1(gk99) worms with an adjusted P-value <0.01. This first list contained 2528 genes (data not shown). We randomly chose 16 genes representing a range of base mean number from 4 to 3500 reads in both or in either N2 or wrn-1 mutant animals based on the RNA-seq normalized data (list of genes in Table 2) to determine the minimum of reads required for a gene to be detected by RT-PCR in our analyses. We thus designed 16 pairs of primers to validate the differential expression of these genes by quantitative RT-PCR. The tubulin encoding gene (tba-1) was included as a control (no difference in tba-1 expression between both strains). PolyA + RNA was thus extracted from three plates of wild type and from three plates of wrn-1(gk99) worms (each plate representing one biological replicate of worms at the L4 stage). The RT-PCR results are shown in Table 2 and the sequences of the primers are provided in the Additional file2: Table S2. Every RT-PCR product was examined on a 2% agarose gel at the end of each run. Figure 2 shows examples of such gels for ten of these genes. A PCR product was detected only in samples that showed more than 145 reads by RNA-seq (Additional file1: Table S1). For example, no band corresponding to nlp-32 mRNA could be detected with the mRNA samples and no Ct could be detected by quantitative RT-PCR. The mean number of reads is below 134 for nlp-32 mRNA in both the wild type and the wrn-1(gk99) worms (Table 2). Similarly, no arrd-24 mRNA could be detected from wild type or wrn-1(gk99) worms mRNA pools even after 40 cycles of PCR (Figure 2). The normalized counts for the arrd-24 gene were below 32 in both genotypes for all biological replicates (Table 2). No Ct could be detected by quantitative RT-PCR. In contrast, a band for the ugt-33 gene was detected with mRNA from the wild type worms only (no band was detected for the wrn-1(gk99) worms) (Figure 2). The mean number of reads was lower than 145 for ugt-33 in the wrn-1(gk99) worms but higher than 145 in the wild type worms (Table 2). Finally, Table 2 indicated expression differences for pes-8, cyp-34a9, cyp-33e2, sodh-1, ugt-33, ech-9, nas-3, gpx-3, acs-2, acs-14, asah-1, lys-8, Y46G5.20, and daf-9 genes between the wild type and the wrn-1(gk99) strains by RT-PCR analyses and such results confirmed the RNA-seq data. A mean number of reads greater than 145 was found in at least one of the genotypes following the RNA-seq analysis for these genes (Table 2). Based on such results, we generated a final list of genes showing at least a two-fold difference in expression between wild type and mutant worms, an adjusted P-value <0.01, and a mean number of reads >145 in at least one of the worm strain. These criteria were applied to each comparison hereafter in this report. Additional file3: Table S3 gives a final list of 1522 genes differentially expressed between wild type and wrn-1(gk99) worms. There were 907 down regulated and 615 up regulated genes respectively (by at least two-fold) in the wrn-1(gk99) worms compared to the wild type worms.Table 1

Bottom Line: We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals.Proteolysis was the only biological process significantly affected by vitamin C in wild type worms.Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche sur le Cancer de l'Université Laval, Hôpital Hôtel-Dieu de Québec (CHU de Québec Research Center), 9 McMahon Sreet, Québec City G1R 2 J6, Canada. michel.lebel@crhdq.ulaval.ca.

ABSTRACT

Background: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase ortholog also exhibits a shorter life span, which can be rescued by vitamin C. In this study, we analyzed the impact of a mutation in the wrn-1 gene and the dietary supplementation of vitamin C on the global mRNA expression of the whole C. elegans by the RNA-seq technology.

Results: Vitamin C increased the mean life span of the wrn-1(gk99) mutant and the N2 wild type strains at 25°C. However, the alteration of gene expression by vitamin C is different between wrn-1(gk99) and wild type strains. We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals. Such genes significantly affected the metabolism of lipid, cellular ketone, organic acid, and carboxylic acids. Vitamin C, in return, altered the expression of genes in wrn-1(gk99) worms involved in locomotion and anatomical structure development. Proteolysis was the only biological process significantly affected by vitamin C in wild type worms.

Conclusions: Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms. Finally, vitamin C extended the life span of wrn-1(gk99) animals by altering biological processes involved mainly in locomotion and anatomical structure development.

Show MeSH
Related in: MedlinePlus