Human DNA Segments Research Articles

Gemogenovatucel-T (Vigil): bi-shRNA plasmid-based targeted immunotherapy.

We describe in this review the historical evidence leading up to the concept and design of Vigil and subsequent clinical applications including safety and efficacy in a randomized, controlled Phase IIB trial. Vigil (gemogenovatucel-T) is a unique triple function targeted immunotherapy that demonstrates preclinical and clinical systemic anticancer activity. Construction of Vigil involves harvest of autologous malignant tissue for neoantigen targeting (ideally containing clonal neoantigens) followed by a two-day process involving transfection with a plasmid to provide a permissive 'training environment' for the patient's immune system. Transfected plasmid components contain an expressive human GMCSF DNA segment to enhance anticancer immune functional response and a second component expressing bi-shRNAfurin which reduces TGFβ isomers (TGFβ1 and TGFβ2) thereby reducing cancer inhibition of the targeted immune response. Results generated to date justify advancement to confirmatory clinical trials supporting product regulatory approval.

The insulin-like growth factor 2 gene in mammals: Organizational complexity within a conserved locus.

The secreted protein, insulin-like growth factor 2 (IGF2), plays a central role in fetal and prenatal growth and development, and is regulated at the genetic level by parental imprinting, being expressed predominantly from the paternally derived chromosome in mice and humans. Here, IGF2/Igf2 and its locus has been examined in 19 mammals from 13 orders spanning ~166 million years of evolutionary development. By using human or mouse DNA segments as queries in genome analyses, and by assessing gene expression using RNA-sequencing libraries, more complexity was identified within IGF2/Igf2 than was annotated previously. Multiple potential 5’ non-coding exons were mapped in most mammals and are presumably linked to distinct IGF2/Igf2 promoters, as shown for several species by interrogating RNA-sequencing libraries. DNA similarity was highest in IGF2/Igf2 coding exons; yet, even though the mature IGF2 protein was conserved, versions of 67 or 70 residues are produced secondary to species-specific maintenance of alternative RNA splicing at a variable intron-exon junction. Adjacent H19 was more divergent than IGF2/Igf2, as expected in a gene for a noncoding RNA, and was identified in only 10/19 species. These results show that common features, including those defining IGF2/Igf2 coding and several non-coding exons, were likely present at the onset of the mammalian radiation, but that others, such as a putative imprinting control region 5’ to H19 and potential enhancer elements 3’ to H19, diversified with speciation. This study also demonstrates that careful analysis of genomic and gene expression repositories can provide new insights into gene structure and regulation.

A Genome Model to Explain Major Features of Neurodevelopmental Disorders in Newborns.

The purpose of this study was to test the hypothesis that infections are linked to chromosomal anomalies that cause neurodevelopmental disorders. In children with disorders in the development of their nervous systems, chromosome anomalies known to cause these disorders were compared with foreign DNAs, including known teratogens. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, cell barriers, structure, epigenetic and chromatin modifications were all found close together in polyfunctional clusters that were deleted or rearranged in neurodevelopmental disorders. In some patients, epigenetic driver mutations also changed access to large chromosome segments. These changes account for immune, circulatory, and structural deficits that accompany neurologic deficits. Specific and repetitive human DNA encompassing large deletions matched infections and passed rigorous artifact tests. Deletions of up to millions of bases accompanied infection-matching sequences and caused massive changes in human homologies to foreign DNAs. In data from 3 independent studies of private, familial, and recurrent chromosomal rearrangements, massive changes in homologous microbiomes were found and may drive rearrangements and encourage pathogens. At least 1 chromosomal anomaly was found to consist of human DNA fragments with a gap that corresponded to a piece of integrated foreign DNA. Microbial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with the epigenome and highly active recombination during meiosis, driven by massive changes in human DNA-foreign DNA homologies. Abnormal recombination in gametes produces zygotes containing rare chromosome anomalies that cause neurologic disorders and nonneurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNAs at a critical stage. Some infections may be more likely tolerated because they resemble human DNA segments. Even rare developmental disorders can be screened for homology to infections within altered epigenomes and chromatin structures. Considering effects of foreign DNAs can assist prenatal and genetic counseling, diagnosis, prevention, and early intervention.

The top 100 most-cited articles on osteosarcoma: a bibliometric analysis

Background: Osteosarcoma or “osteogenic sarcoma” is the most common neoplasm of the bone in children and young adults. This research is premised upon a citation analysis of the top 100 most-cited articles on osteosarcoma. Materials and Methods: This research is dependent upon the use of SCOPUS database. Using the search strategy “osteosarcoma” on this database yielded 12,107 articles. After filtering for relevant articles, the top 100 most-cited articles were retrieved for descriptive and statistical analysis. Results: The most-cited paper was “A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma” by Friend and colleagues published in 1986 with 1888 citations. The publication years ranged from 1967 to 2014. The United States was the most productive country in terms of research output, followed by Italy. Institution-wise, the Memorial Sloan-Kettering Cancer Centre contributed the most articles. However, the most prolific author was from Italy. Majority of the publications were level IV studies Conclusions: Although citation analysis is not entirely flawless, this is a comprehensive list of the top 100 most-cited articles significantly impacting osteosarcoma knowledge and research over time. Future studies need to cater toward maximizing high-quality evidenced articles.

Sequence-Dependent Persistence Length of Long DNA.

Using a high-throughput genome-mapping approach, we obtained circa 50million measurements of the extension of internal human DNA segments in a 41 nm×41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5-393kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk's theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.

Learning from the Uncontrollable

Research papers are usually revisionist histories, imagined accounts driven by impeccable logic and unbroken experimental success, which in no way reflect the slowness, messiness, and serendipity of real life in the lab. To give a different hopefully more balanced perspective than what is often told, I want to tell you the story behind some of the discoveries that shaped my early career.“In reality, of course, I had stumbled on the control by complete accident.”

The effects of monovalent metal ions on the conformation of human telomere DNA using analytical ultracentrifugation.

A human telomere DNA segment (HT-DNA) can fold into a G-quadruplex in the presence of some monovalent cations. These cations can interact with the phosphate groups of the DNA segment and/or with the O6 oxygen atom of guanines, which are called non-specific interactions and specific interactions, respectively. However, until now how these two interactions affect the structure of HT-DNA has not been well understood. In this study, a combination of analytical ultracentrifugation (AUC) and circular dichroism (CD) was used to explore the effects of these two interactions on the structure of a 22-mer single-stranded DNA with a sequence of 5'-AGGG(TTAGGG)3-3'. The results showed that the standard sedimentation coefficient (s20,w) of HT-DNA starts to increase when the concentration of potassium ions (CK(+)) is higher than 10.0 µM due to the formation of a G-quadruplex through specific interactions. Whereas, for a control DNA, a higher CK(+) value of 1.0 mM was needed for increasing s20,w due to non-specific interactions. Moreover, potassium ions could promote the formation of the G-quadruplex much more easily than lithium, sodium and cesium ions, presumably due to its appropriate size in the dehydrated state and easier dehydration. The molar mass of DNA at different cation concentrations was nearly a constant and close to the theoretical value of the molar mass of monomeric HT-DNA, indicating that what we observed is the structural change of individual DNA chains.

Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

Abstract A06: Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome

Abstract Our interests lie in humanizing specific regions of the mouse genome using large segments of human DNA with extents of 10s to 100s of kilobase pairs. To that end, we have undertaken a series of projects to transfer large segments of human DNA, from human BAC clones, into the orthologous region of corresponding mouse BAC clones using— (1), traditional recombineering methods; (2), recombinase-mediated cassette exchange (RCME); and (3), a novel technology that we call trans- or trimolecular-recombineering. In each instance the goal is to place human DNA within the larger context of homologous BAC-derived mouse arms in E. coli. Completed constructs will be used to drive homologous recombination in mouse embryonic stem (ES) cells with positive selection imparted at each end of the transferred human segment using dual, recombinase recognition site-flanked, selectable marker cassettes. Properly targeted ES cells will be used to develop viable mouse strains carrying the humanized segments. Selection cassettes will be removed by crossing to mouse strains with germline expression of the appropriate recombinases. All experiments have been designed to maintain a fixed genetic background from community-standard C57BL/6 strains. Our poster presentation details the specifics of our projects and describes progress to date. This research is supported by National Cancer Institute Grant Number 5P30CA034196-28. Citation Format: Tiffany Leidy-Davis, David E. Bergstrom. Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A06.

MiRNA nano-packages for maternal-placental-fetal communication

Positioned at the maternal-fetal interface, the placenta generates regulatory signals and serves as a communication junction that governs pregnancy health. In addition to hormones, growth factors and diverse proteins, we and others have recently shown that small non-coding RNA molecules, including microRNAs (miRNAs), traffic among the maternalplacental-fetal compartments, and may have a central role in maternalfetal adaptation to homeostatic perturbations. Although common and unique types of miRNAs are expressed by the placenta during pregnancy, the function of placental miRNA species remains largely unknown. We recently showed that primary human trophoblasts are resistant to infection by a heterogeneous panel of viruses, and that this resistance can be partly conferred to non-trophoblast cells by exogenous expression of trophoblast-specific miRNAs, expressed from the chromosome 19 miRNA cluster (C19MC). This effect was not observed during cell infection by nonviral placental pathogens, including Listeria monocytogenes, and Toxoplasma gondii. Trophoblastic miRNAs can be released to the extracellular space within several types of vesicles, including cell fragments, apoptotic bodies, microvesicles, and nanovesicles (exosomes), or can be packaged with non-vesicular protein complexes, such as argonaute-2. To examine the trafficking of trophoblastic miRNAs to maternal-fetal tissues we engineered a transgenic mouse that expresses a 160 kb segment of human genomic DNA harboring the human C19MC cluster. We coupled this technology with RNAseq analysis of miRNA expression in triads of placenta, maternal plasma and fetal plasma from pregnant women at term. Together, these analyses shed new light on transplacental bidirectional miRNA trafficking patterns (Supported by NIH HD06589, HD075665, HD071707, AI081759, and the Pennsylvania Department of Health Research Formula Funds)

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine β-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

Feeling Funk-y: Human Gene Patents in AMP v. Myriad

In a stunning reversal of the long-standing practice of the U.S. Patent & Trademark Office permitting the patenting of human DNA, the Supreme Court unanimously held on June 13, 2013, in Association for Molecular Pathology v. Myriad Genetics, Inc., that an isolated segment of human DNA, such as a gene, is not eligible for patent protection under 35 USC x101. The decision suggests that more human intervention beyond mere cleaving of this naturally occurring molecule from its natural environment is required. The Court did not prevent patent protection on all genetic materials, however, as it also held that a type of nonnaturally occurring molecule known as complementary DNA was patent eligible. The Court’s analysis of these DNA-based claims was guided by two precedents, Funk Brothers Seed Co. v. Kalo Inoculant Co. and Diamond v. Chakrabarty, which addressed the patent eligibility of biological organisms. In this article, we examine these two precedents, how the Supreme Court applied them in its decision in Myriad, and some potential implications of the Myriad decision.

Increased genome instability in human DNA segments with self-chains: homology-induced structural variations via replicative mechanisms

Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that 'self-chain' (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

The structural complexity of DNA templates—Implications on cellular complexity

A previously formulated procedure for the quantitative evaluation of the complexities of molecules and biostructures is applied to assess the complexities of selected genomic DNA sequences. These include: (1) Several E. coli genes, including lacI, as examples of DNA sequences which are nearly as complex as possible (relative complexity=∼1). This is verified by the Lempel–Ziv (LZ) complexity analysis. (2) The telomere of a yeast chromosome, which has a considerable number of regular features that reduce complexity; the telomere shows indeed a lower structural complexity value. (3) A segment of human DNA, gene p53, which has a certain number of regular features such as 29 interspersed alu elements; these features cause a certain reduction in the complexity of the p53 gene, but do not invalidate the (previous) overall conclusion that template complexity is very high. The close to maximal complexity of the transcribed regions of p53 is validated by the LZ compression analysis. The general conclusion is that DNA base sequence composition is the dominant factor determining cellular complexity. The high complexity of DNA arrived at is a direct consequence of the template character of DNA and reflects the role of genomic DNA as a principal regulating element of a cell. It will be a challenge to find systems of lower complexity with the ability to respond to challenges from the environment to the extent that DNA templated systems do. Cellular complexity and template directed activity are thus highly intertwined properties, at the heart of many developmental, behavioral and evolutionary processes.

Common fragile sites are conserved features of human and mouse chromosomes and relate to large active genes

Common fragile sites (CFSs) are seen as chromosomal gaps and breaks brought about by inhibition of replication, and it is thought that they cluster with tumor breakpoints. This study presents a comprehensive analysis using conventional and molecular cytogenetic mapping of CFSs and their expression frequencies in two mouse strains, BALB/c and C57BL/6, and in human probands. Here we show that induced mouse CFSs relate to sites of spontaneous gaps and breaks and that CFS expression levels in chromosome bands are conserved between the two mouse strains and between syntenic mouse and human DNA segments. Furthermore, four additional mouse CFSs were found to be homologous to human CFSs on the molecular cytogenetic level (Fra2D-FRA2G, Fra4C2-FRA9E, Fra6A3.1-FRA7G, and Fra6B1-FRA7H), increasing the number of such CFSs already described in the literature to eight. Contrary to previous reports, DNA helix flexibility is not increased in the 15 human and eight mouse CFSs molecularly defined so far, compared to large nonfragile control regions. Our findings suggest that the mechanisms that provoke instability at CFSs are evolutionarily conserved. The role that large transcriptionally active genes may play in CFS expression is discussed.

Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast Saccharomyces cerevisiae.

The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.

The diabetes-prone BB rat carries a frameshift mutation in Ian4, a positional candidate of Iddm1.

Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling human type 1 diabetes. They also exhibit lifelong T-cell lymphopenia. Functional and genetic data support the hypothesis that the gene responsible for the lymphopenia, Lyp, is also a diabetes susceptibility gene, named Iddm1. We constructed a 550-kb P1-derived artificial chromosome contig of the region. Here, we present a corrected genetic map reducing the genetic interval to 0.2 cM and the physical interval to 150-290 kb. A total of 13 genes and six GenomeScan models are assigned to the homologous human DNA segment on HSA7q36.1, 8 of which belong to the family of immune-associated nucleotides (Ian genes). Two of these are orthologous to mouse Ian1 and -4, both excellent candidates for Iddm1. In normal rats, they are expressed in the thymus and T-cell regions of the spleen. In the thymus of lymphopenic rats, Ian1 exhibits wild-type expression patterns, whereas Ian4 expression is reduced. Mutational screening of their coding sequences revealed a frameshift mutation in Ian4 among lymphopenic rats. The mutation results in a truncated protein in which the COOH-terminal 215 amino acids-including the anchor localizing the protein to the outer mitochondrial membrane-are replaced by 19 other amino acids. We propose that Ian4 is identical to Iddm1.

Origin of caucasoid-specific mitochondrial DNA lineages in the ethnic populations of the Altai-Sayan region

The data on sequence variation in the first hypervariable segment (HVSI) of human mitochondrial DNA (mtDNA) representing Caucasoid mtDNA lineages in the gene pools of Altaians and Khakassians are presented. Identification of the subgroups of Caucasoid mtDNA lineages found in the gene pools of the ethnic populations of the Altai-Sayan region and the adjacent territories, Altaians, Khakassians, Tuvinians, Buryats, and Yakuts was carried out. All Caucasoid mtDNA lineages belonged to groups H, HV1, J*, J1, J1b1, T1, T4, U1a, U2, U3, U4, U5a1, I, X and N1a. Taking into consideration possible contribution of southern Caucasoid and eastern European components to the formation of the anthropological type of Altai-Sayan ethnic populations, distribution of the revealed Caucasoid mtDNA lineages among the ethnic populations of the Central Asia, Western Asia, Caucasus, and Eastern Europe was examined. The applied approach permitted identification of 60% of mtDNA types the majority of which had southern Caucasoid origin. Less than 10% of mtDNA types were of eastern European origin. The gene pools of Altaians and Khakassians displayed the presence of autochthonous components represented by mtDNA types from subgroups U2 and U4.

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR

Proper cellular response to genotoxic insult often requires the activity of one or more members of a family of high-molecular weight protein kinases referred to as phosphatidylinositol-3 kinase (PIK)-like proteins. While catalytic activity is an indispensable part of PIK-like protein function, little is currently known about factors that control their activity and/or functions. This deficiency stems, in large part, from our lack of knowledge concerning functionally significant subdomains within the large non-catalytic domain of these proteins. We have determined that the transcript encoding the PIK-like protein ATR undergoes alternate splicing within the region of the mRNA encoding its non-catalytic domain. This conclusion is based on the sequencing of a human expressed sequence tag clone encoding a portion of the ATR cDNA, and is supported by the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays conducted on total and polyA+ RNA, as well as sequencing of cloned RT-PCR products. Cloning and sequencing of a segment of human genomic DNA indicated that this event arises from splicing of a single 192 bp exon within the ATR gene. Analysis of several human tissues indicated that alternate ATR transcripts are differentially expressed, suggesting that this region of the ATR protein may be of functional importance.

Characterization of Human Junctophilin Subtype Genes

Junctophilin (JP) subtypes, namely JP-1, 2, and 3, have been currently identified in excitable cells and constitute a novel family of junctional membrane complex proteins. Our studies have suggested that JPs take part in the formation of junctional membrane complexes by spanning the membrane of the intracellular Ca2+ store and interacting with the cell-surface membrane. In this report we describe the primary structures, genomic organization, and tissue distribution of human JP subtypes. By cloning and analyzing human genomic DNA segments, the protein-coding sequence interrupted with four introns was defined in each JP gene. The deduced human JP subtypes shared characteristic structural features with their rabbit and mouse counterparts. Genomic mapping demonstrated that JP genes do not cluster on the human genome. RNA blot hybridization indicated that tissue-specific expression patterns of JP genes in human are essentially the same as those in mouse; skeletal muscle contained both JP-1 and JP-2 mRNAs, the heart predominantly expressed JP-2 mRNA, and the brain specifically contained JP-3 mRNA. In the light of this, we propose intramolecular domains of JP subtypes based on the structural and functional characteristics.