Downregulation Of 14q32 Micrornas In Primary Human Desmoplastic Medulloblastoma

Medulloblastoma (MB) is one of the most common pediatric cancers, likely originating from abnormal development of cerebellar progenitor neurons. MicroRNA (miRNA) has been shown to play an important role in the development of the central nervous system. Microarray analysis was used to investigate miRNA expression in desmoplastic MB from patients diagnosed at a young age (1 or 2 years old). Normal fetal or newborn cerebellum was used as control. A total of 84 differentially expressed miRNAs (64 downregulated and 20 upregulated) were found. Most downregulated miRNAs (32/64) were found to belong to the cluster of miRNAs at the 14q32 locus, suggesting that this miRNA locus is regulated as a module in MB. Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. First, expression of estrogen-related receptor-γ (ESRRG), a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. Second, expression of the parentally imprinted gene MEG3 was lower in MB in comparison to normal cerebellum, suggesting a possible epigenetic silencing of the 14q32 locus. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2), and miR-323-3p (14q32.2), were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. 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Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<US$10) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a ß-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<US$10) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a ß-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality

The Role Of The Opaque2 Transcriptional Factor In The Regulation Of Protein Accumulation And Amino Acid Metabolism In Maize Seeds.

[No abstract available]66 Su 1Pt 2

The Involvement Of Opaque 2 On β-prolamin Gene Regulation In Maize And Coix Suggests A More General Role For This Transcriptional Activator

The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa α-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa α-coixin gene family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the β-prolamins. A new O2-binding box was identified in β-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived. © 1995 Kluwer Academic Publishers.2751015102

Structural Characterization And Promoter Activity Analysis Of The γ-kafirin Gene From Sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions -192 and -476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response. © 1994 Springer-Verlag.245217718

Sequence Analysis Of 22 Kda-like α-coixin Genes And Their Comparison With Homologous Zein And Kafirin Genes Reveals Highly Conserved Protein Structure And Regulatory Elements

Several genomic and cDNA clones encoding the 22 kDa-like α-coixin, the α-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like α-coixin genes designated α-3A, α-3B and α-3C were found in the 15 kb α-3 genomic clone. The α-3A and α-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the α-3B gene, suggesting that the three α-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of α-coixin clones with the published sequences of 22 kDa α-zein and 22 kDa-like α-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like α-prolamins and the 19 kDa α-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5′ and 3′ flanking regions of α-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in α-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in α-3B that occupies approximately the same positions as those identified for the 22 kDa α-zein and α-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like α-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation. © 1993 Kluwer Academic Publishers.21576577