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PubMed1982

Complexity of polysomal polyadenylated RNAs in Vicia faba meristematic root cells.

As a first step in quantifying gene expression during differentiation of the Vicia faba root cells we have analysed the mRNA population from meristematic cells. Using cDNA X mRNA hybridizations we have shown that this population can be divided into three abundance classes (abundant, intermediate and rate) representing 37%, 34% and 29% of the mRNAs and containing 26, 610 and 11700 sequences respectively. The total base-sequence complexity of the mRNA population, as determined by cDNA X mRNA hybridization, was found to be 1.6 x 10(7) nucleotides. This estimate was confirmed by the determination of the amount of single-copy genomic DNA hybridizing to the mRNA. Expressed as the number of structural gene transcripts, this complexity corresponds to about 13000 mRNA sequences.

DNA↗
PubMed1999

The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae.

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.

Acetyltransferases↗
PubMed1998

Gain of DNA copy number on chromosomes 3q26-qter and 5p14-pter is a frequent finding in head and neck squamous cell carcinomas.

Comparative genomic hybridization (CGH) was used to study genomic imbalances in 77 squamous cell carcinomas of the head and neck (HNSCC) and in four cell lines derived from oral carcinomas. Particular attention was paid to all tumors characterized by a gain of two specific chromosomal segments, i.e. 3q26-qter and 5p14-p15. The 57 tumors containing both or one of the two imbalances were compared with 20 tumors lacking both with regard to genomic complexity, as well as to associated genomic, histopathologic and clinical peculiarities. 60% of the oral, and 66% of the non-oral cancers exhibited a gain of 3q26-qter, while a gain of 5p14-p15 was found in 66% of the oral, but only in 48% of the non-oral tumors. 48% of all tumors were characterized by both gains together, 26% exhibited only one of the two alterations. It could be shown that presence of both, gain of 3q26-qter and 5p14-p15, was clearly associated with a significantly higher complexity of genomic changes which was not only expressed as a high frequency of DNA copy number alterations (CNAs) but was also connected with a considerable number of additional amplifications of various chromosomal segments and a high conformity of the patterns of genomic imbalances in these tumors. Clear differences of the extent and of the patterns of genomic imbalance could be observed between oral and non-oral tumors. With respect to histopathological parameters, no clear association could be found for specific imbalances to the grade of differentiation, nor the invasiveness or metastatic status of the tumors. However, a higher number of patients with tumors characterized by gain of 3q26-qter plus 5p14-p15 died within a short period (i.e. less than 15 months) after excision of the tumor compared to the group without these imbalances. The implications of these findings are discussed from the oncogenetic and clinical aspects and in comparison with other reports.

Journal Article↗
PubMed1996

High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.

Animals↗
PubMed2001

Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Animals↗
PubMed2026

Long-read based detection of large copy number variants with potential functional significance using the ContextSV structural variant caller.

Long-read sequencing enables improved detection of structural variants (SVs) in the human genome due to its substantially increased read lengths. However, currently widely used long-read SV callers primarily rely on alignment-based evidence, limiting their ability to detect large and complex SVs and potentially missing disease-relevant events. To address these limitations, we developed ContextSV, a framework that integrates alignment evidence with copy number predictions derived from sequencing coverage and single-nucleotide variant allele frequencies to improve SV detection, particularly for large copy number variants (CNVs). We additionally developed ContextScore, a machine learning-based classification model to assign SV confidence scores based on genomic context features and integrated it within ContextSV. Through benchmarking analyses on both simulated and real datasets, we demonstrate that ContextSV improves detection of large CNVs and inversions that may be missed by existing long-read SV callers. We further illustrate its utility by identifying and experimentally validating multiple large SVs in the KOLF2.1J reference stem cell line that were not detected by other methods. Collectively, our results demonstrate that ContextSV serves as a valuable complement to existing long-read SV detection approaches by improving sensitivity for large and clinically relevant SVs.

Humans↗
PubMed2001

Plasmid transcriptional repressor CopG oligomerises to render helical superstructures unbound and in complexes with oligonucleotides.

CopG is a 45 amino acid residue transcriptional repressor involved in the copy number control of the streptococcal plasmid pMV158. To do so, it binds to a DNA operator that contains a 13 bp pseudosymmetric DNA element. Binding of CopG to its operator results in repression, at the transcriptional level, of its own synthesis and that of the initiator of replication protein, RepB. Biochemical experiments have shown that CopG co-operatively associates to its target DNA at low protein:DNA ratios, completely protecting four helical turns on the same face of the double helix in both directions from the inverted repeat that constitutes the CopG primary target. This has been correlated with a CopG-mediated DNA bend of about 100 degrees. Here, we show that binding of CopG to DNA fragments containing the inverted repeat just at one end led to nucleation of the protein initiating from the inverted repeat. Nucleation extended to the entire fragment, with CopG-DNA contacts occurring on the same face of the DNA helix. The protein, the prototype for a family of homologous plasmid repressors, displays a homodimeric ribbon-helix-helix arrangement. It polymerises within the unbound crystal to render a continuous right-handed protein superhelix of homodimers, around which a bound double-stranded (ds) DNA could wrap. We have solved the crystal structure of CopG in complex with a 22 bp dsDNA oligonucleotide encompassing the cognate pseudosymmetric element. In the crystal, one protein tetramer binds at one face of the DNA with two parallel beta-ribbons inserted into the major groove. The DNA is bent about 50 degrees under compression of both major and minor grooves. A continuous right-handed complex helix made up mainly by protein-protein and some protein-DNA interactions is observed. The protein-protein interactions involve regions similar to those observed in the oligomerisation of the native crystals and those employed to set up the functional tetramer. A previously solved complex structure of the protein with a 19 bp dsDNA had unveiled a left-handed helical superstructure just made up by DNA interactions.

Bacterial Proteins↗
PubMed1999

Recurrent DNA copy number losses associated with metastasis of larynx carcinoma.

Squamous cell carcinomas of the head and neck show frequent and complex chromosome aberrations, but little is known about the changes that occur during the metastatic process. To compare the accumulation of changes in primary and metastatic tumors we analyzed 19 pairs of primary larynx cancer tumors and their metastases. The most frequent changes were found at 3p, 3q, 5p, 9, and 13. Losses at 13, 8p, and 9q were more frequent in metastases than in primary tumors.

Adult↗
PubMed1992

Relevance of the quantitative detection of HIV proviral sequences in PBMC of infected individuals.

Changes in HIV replication during progression of HIV infection were assessed by estimation of the number of peripheral blood mononuclear cells (PBMC) harboring HIV proviral sequences. Samples from 23 patients at different stages of HIV infection were analyzed by quantitative polymerase chain reaction (PCR) using GAG primers. HIV titers in PBMC were also determined by serial dilutions of cells in coculture with phytohemagglutinin-activated normal PBMC. A positive correlation was observed between the number of HIV DNA copies and the HIV titer in PBMC. The PCR test was more sensitive than the coculture technique. The number of HIV copies detected by PCR ranged from 50 to 10,500 per 10(6) PBMC: assuming one copy per cell this implies a frequency of proviral HIV-containing cells of one per 100 to one per 20,000 mononuclear cells. The mean number of HIV DNA sequences in PBMC was significantly lower in asymptomatic patients than in AIDS patients and patients with AIDS-related complex (ARC). In patients who progressed from asymptomatic infection to AIDS, the number of HIV DNA copies in PBMC rose, indicating an increase of HIV replication. These results show that the number of infected PBMC increases during clinical progression. However, some asymptomatic patients had a higher number of HIV DNA copies in their PBMCs suggesting that increased HIV replication precedes the appearance of clinical symptoms.

DNA, Viral↗
PubMed2003

End-sequence profiling: sequence-based analysis of aberrant genomes.

Genome rearrangements are important in evolution, cancer, and other diseases. Precise mapping of the rearrangements is essential for identification of the involved genes, and many techniques have been developed for this purpose. We show here that end-sequence profiling (ESP) is particularly well suited to this purpose. ESP is accomplished by constructing a bacterial artificial chromosome (BAC) library from a test genome, measuring BAC end sequences, and mapping end-sequence pairs onto the normal genome sequence. Plots of BAC end-sequences density identify copy number abnormalities at high resolution. BACs spanning structural aberrations have end pairs that map abnormally far apart on the normal genome sequence. These pairs can then be sequenced to determine the involved genes and breakpoint sequences. ESP analysis of the breast cancer cell line MCF-7 demonstrated its utility for analysis of complex genomes. End sequencing of approximately 8,000 clones (0.37-fold haploid genome clonal coverage) produced a comprehensive genome copy number map of the MCF-7 genome at better than 300-kb resolution and identified 381 genome breakpoints, a subset of which was verified by fluorescence in situ hybridization mapping and sequencing.

Animals↗
PubMed2006

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Algorithms↗
PubMed1992

Prospects for the genetic engineering of milk.

Milk and milk products comprise a substantial fraction of the protein intake of the industrialised West. The establishment of germline manipulation techniques in cows offers opportunities for directly manipulating milk composition to produce products with enhanced nutritional and processing properties. The major milk proteins are encoded by a small number of abundantly expressed single-copy genes and a number of possible manipulations are described. Milk proteins exhibit complex interactions with each other and with other constituents of milk. It will, therefore, be necessary to utilise model systems to evaluate the consequences of these proposed changes before embarking upon the costly and time-consuming process of manipulating the bovine genome.

Animals↗
PubMedDate not supplied

Congenital heart defects in siblings with partial aneuploidy of chromosome 2p.

Cytogenetic anomalies are found in 13% of children with congenital heart disease, providing positional information that may aid in identifying genes governing cardiac development. We report two children born to a clinically normal female with a balanced chromosomal rearrangement, resulting from insertion of 2p24.2-p25.1 into chromosome 1. Each of her offspring had complex congenital heart disease, including left ventricular hypoplasia, and inherited a different copy number of the 2p segment. These findings suggest that proper dosage of a gene or genes in this region is important in normal cardiac development.

Aneuploidy↗
PubMed1996

Gene identification in a complex chromosomal continuum by local genomic cross-referencing.

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.

Alcohol Dehydrogenase↗
PubMed2026

Using cancer profiles to identify synthetic lethal therapeutic targets and predictive biomarkers in cancer gene dependency data.

MOTIVATION: Large scale loss-of-function screens utilising CRISPR or siRNA can provide profound insights into the importance of individual genes for the survival of a cancer cell and can drive the identification of therapeutic targets and biomarkers, and the development of targeted drugs. However, the analysis of these data and the substantial bodies of metadata that relate to them, is technically challenging and typically requires substantial expertise in data science and computer coding. RESULTS: To facilitate the analysis of cancer gene dependency data by cancer biologists and clinical scientists, we have developed DepMine-a computational toolkit providing a powerful system for framing complex queries relating cancer gene dependency to the underlying genetic changes that occur in cancer cells. DepMine identifies synthetic lethal relationships between putative target genes and complex 'cancer profiles' built from user-specified combinations of mutations, copy-number variation, and expression levels, and can refine these to optimal biomarker definitions for target dependency. AVAILABILITY: The Python implementation of DepMine and associated data files can be obtained at https://github.com/UOSbioinformaticslab/depmine and is free to academics and Not-For-Profit organisations. The DepMine release referenced in this paper is archived as DOI: 10.5281/zenodo.19570601.

Humans↗
PubMed1990

Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes.

When microinjected foreign genes integrate into the genomes of mice, multiple copies are frequently found clustered together at one location. How they concatamerize--by the integration of large linearized concatamers that are formed by simple end-to-end linkage, by circularization of individual DNA fragments and recombination, or by some other means--is not understood. In the transgenic animals studied thus far by ourselves and others, integration frequency and transgene copy number do not seem to be significantly influenced by the complementarity of the ends of the DNA fragments that have been microinjected. We have utilized PCR amplification and DNA sequence analysis to study selected transgene junctions at the nucleotide level. In two transgenic mice carrying the synthetic RSVcat gene (injected with noncomplementary overhangs on the fragment ends), ends were 'nibbled' from 1 to 62 bases before being joined to an adjacent gene copy. Repeated dinucleotides, providing the most minimal of homologies, are present in half of the characterized junctions. Determination of the relative copy number of the junctions in each mouse supports the idea that transgene complexes can undergo additional rearrangements after the initial formation event.

Animals↗
PubMed2004

Gene expression from the aneuploid chromosome in a trisomy mouse model of down syndrome.

Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the approximately 230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number.

Aneuploidy↗
PubMed1992

DNA probes for the identification of members of the Simulium damnosum complex (Diptera: Simuliidae).

Genomic libraries in plasmid have been constructed from various sibling species of blackflies of the Simulium damnosum complex from West Africa. Three cloned repetitive sequences, which show variation in copy number between sibling species, have been isolated. These clones can be used as probes for the dot-blot identification of larvae, pupae or adults into the three main West African subcomplexes, i.e. damnosum, squamosum and sanctipauli subcomplexes. The sequences also show some intraspecific variation in copy number.

Animals↗