The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source ...of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings.
We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs.
By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.
Nanotechnology has considerable promise for the detection, staging and treatment of cancer. Here, we outline one such promising application: the use of nanostructures with surface-bound ligands for ...the targeted delivery and ablation of colorectal cancer (CRC), the third most common malignancy and the second most common cause of cancer-related mortality in the US. Normal colonic epithelial cells as well as primary CRC and metastatic tumors all express a unique surface-bound guanylyl cyclase C (GCC), which binds the diarrheagenic bacterial heat-stable peptide enterotoxin ST. This makes GCC a potential target for metastatic tumor ablation using ST-bound nanoparticles in combination with thermal ablation with near-infrared or radiofrequency energy absorption. Furthermore, the incorporation of iron or iron oxide into such structures would provide advantages for magnetic resonance imaging (MRI). Although the scenarios outlined in this article are hypothetical, they might stimulate ideas about how other cancers could be attacked using nanotechnology.
Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt ...bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.
Hearing impairment affects one infant in 1000 and 4% of people aged younger than 45 years. Congenital deafness is inherited or apparently sporadic. We have shown previously that
DFNB1 on chromosome ...13 is a major locus for recessive deafness in about 80% of Mediterranean families and that the connexin-26 gene gap junction protein β2 (
GJB2) is mutated in
DFNB1 families. We investigated mutations in the
GJB2 gene in familial and sporadic cases of deafness.
We obtained DNA samples from 82 families from Italy and Spain with recessive non-syndromic deafness and from 54 unrelated participants with apparently sporadic congenital deafness. We analysed the coding region of the
GJB2 gene for mutations. We also tested 280 unrelated people from the general populations of Italy and Spain for the frameshift mutation 35delG.
49% of participants with recessive deafness and 37% of sporadic cases had mutations in the
GJB2 gene. The 35delG mutation accounted for 85% of
GJB2 mutations, six other mutations accounted for 6% of alleles, and no changes in the coding region of
GJB2 were detected in 9% of
DFNB1 alleles. The carrier frequency of mutation 35delG among people from the general population was one in 31 (95% CI one in 19 to one in 87).
Mutations in the
GJB2 gene are a major cause of inherited and apparently sporadic congenital deafness. Mutation 35delG is the most common mutation for sensorineural deafness. Identification of 35delG and other mutations in the
GJB2 gene should facilitate diagnosis and counselling for the most common genetic form of deafness.
Silicon-based chips with discrete, independently temperature-controlled islands have been developed for use in DNA microarray hybridization studies. Each island, containing a heater made of a ...diffusion layer and a temperature sensor based on a p-n junction, is created on a silicon dioxide/nitride surface by anisotropic etching. Different reactive groups are subsequently added to the surface of the islands, and allele-specific oligonucleotide probes are attached to discrete spots on the chip. Hybridization is performed with Cy5-tagged single-stranded targets derived by PCR from genomic DNA. Results are assessed by measuring fluorescence of bound dye-tagged targets after hybridization and washing. Temperatures at each island can be set at different values to obtain optimal distinction between perfect matches and mismatches. This approach facilitates definition of optimal temperatures for probe/target annealing and for distinction between perfectly matched versus mismatched solution-phase targets. The thermal gradient DNA chips were then tested for genotyping, and the results for four different loci in two genes are presented. Unambiguous typing was achieved for clinically relevant loci within the factor VII and hemochromatosis genes.
Understanding regulation of fetal and embryonic hemoglobin expression is critical, since their expression decreases clinical severity in sickle cell disease and beta-thalassemia. K562 cells, a human ...erythroleukemia cell line, can differentiate along erythroid or megakaryocytic lineages and serve as a model for regulation of fetal/embryonic globin expression. We used microarray expression profiling to characterize transcriptomes from K562 cells treated for various times with hemin, an inducer of erythroid commitment. Approximately 5,000 genes were expressed irrespective of treatment. Comparative expression analysis (CEA) identified 899 genes as differentially expressed; analysis by the self-organizing map (SOM) algorithm clustered 425 genes into 8 distinct expression patterns, 322 of which were shared by both analyses. Differential expression of a subset of genes was validated by real-time RT-PCR. Analysis of 5'-flanking regions from differentially expressed genes by PAINT v3.0 software showed enrichment in specific transcription regulatory elements (TREs), some localizing to different expression clusters. This finding suggests coordinate regulation of cluster members by specific TREs. Finally, our findings provide new insights into rate-limiting steps in the appearance of heme-containing hemoglobin tetramers in these cells.
The ubiquitin/proteasome pathway for degradation of completed and nascent globin chains was evaluated using a cell-free in vitro coupled transcription/translation assay. No decrease in radiolabeled ...globin chains was observed when ubiquitin, energy regenerating
source (or ATP), and E1 and E2 enzymes were added 30 min after the start of translation when globin chain synthesis had plateaued.
In contrast, the addition of these components prior to the start of translation resulted in no radiolabeled globin chains
after 30 min. The loss of radiolabeled globin chains was dependent on ATP concentration; the higher the concentration, the
less the radiolabeled globin chains formed. Prior to the initiation of transcription/translation, cell extract was preincubated
with the proteasomal inhibitor MG132 in the absence of globin chain expression vector after which ubiquitin-protein isopeptidase
inhibitor, Ubal, and expression vector were added in the presence of 1.5 m m ATP. Thereafter, radiolabeled monoubiquitylated and multiubiquitylated globin chains with few unmodified globin chains were
formed. Our results suggest that polyubiquitylated globin chains are localized to the polysomal fractions. These results suggest
that nascent globin chains are potential targets for ubiquitylation and deubiquitylation during or soon after translation
and that ATP levels play a role in the balance between polypeptide synthesis and degradation.
Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.
Primer ...pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.
Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.
The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.
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