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PubMed2006

Engineering embryonic stem cells with recombinase systems.

The combined use of site-specific recombination and gene targeting or trapping in embryonic stem cells (ESCs) has resulted in the emergence of technologies that enable the induction of mouse mutations in a prespecified temporal and spatially restricted manner. Their large-scale implementation by several international mouse mutagenesis programs will lead to the assembly of a library of ES cell lines harboring conditional mutations in every single gene of the mouse genome. In anticipation of this unprecedented resource, this chapter will focus on site-specific recombination strategies and issues pertinent to ESCs and mice. The upcoming ESC resource and the increasing sophistication of site-specific recombination technologies will greatly assist the functional annotation of the human genome and the animal modeling of human disease.

Animals↗
PubMed1987

Region-specific expression of mouse homeobox genes in the embryonic mesoderm and central nervous system.

The homeobox is a 180-base-pair sequence characteristically found in homeotic and segmentation genes in Drosophila. Several copies of homeoboxes are also found in the mammalian genome, but it is not known whether these are components of morphogenetic loci in mammals as well. As a step toward understanding the function of mammalian homeoboxes, we have used in situ hybridization to define the spatial pattern of expression of two mouse homeobox genes in the midgestational mouse embryo. The two mouse homeoboxes studied here, Hox 1.2 and Hox 1.4, are located 20 kilobases apart on mouse chromosome 6. Our results demonstrate the following: (i) Hox 1.2 transcripts are localized mainly in the posterior myelencephalon, in the cervical central nervous system (CNS), and in several thoracic prevertebrae; (ii) Hox 1.4 transcripts are localized mainly in the posterior myelencephalon and in the cervical CNS; (iii) within the CNS region expressing Hox 1.4, the level of Hox 1.4 transcripts is higher in the mantle layer than in the ependymal layer and higher in the dorsal than in the ventral area. The specific localization of Hox 1.2 and Hox 1.4 transcripts in the embryonic CNS and the restricted pattern of expression along the rostrocaudal axis are strikingly reminiscent of the expression pattern of Drosophila homeoboxes in the fly embryo and larvae. Despite the different developmental strategies adopted by Drosophila and mammals, functional similarities may exist between Drosophila and mammalian homeobox genes.

Animals↗
PubMed2026

Multinucleated Giant Cells in Human Pancreatic Cancer Are a Distinct Macrophage Population Undergoing a DNA Damage Response and Associated with an Aggressive Tumor Microenvironment.

Macrophages (Mϕ) constitute a dominant and functionally diverse immune population within the microenvironment of pancreatic ductal adenocarcinoma (PDAC), yet how Mϕ heterogeneity contributes to the tumor remains poorly defined. In an institutional cohort of 145 PDAC specimens, we identified a population of multinucleated giant cells (MGC) of Mϕ origin, an entity previously described in chronic inflammation but rarely in cancer. CD68+ MGCs were present in 28% of tumors, enriched in squamous, nonglandular regions, and more frequent after neoadjuvant chemotherapy. By integrating spatial transcriptomics and quantitative imaging, we defined the features of these cells, which, compared with MGCs in nonneoplastic inflammatory lesions, lacked canonical polarization markers (HLA-DR and CD163) and displayed a distinctive transcriptional program characterized by upregulation of the POLR2K, TUBA8, COX5B, and VDAC1 genes, which encode proteins involved in DNA repair, oxidative stress, and MYC signaling. Spatial analyses revealed activation of hypoxia and extracellular matrix-remodeling pathways in MGC-associated niches, and experimental hypoxia promoted MGC formation in vitro. Consistent with these data, we found that in the The Cancer Genome Atlas (TCGA) Pancreatic Adenocarcinoma (PAAD) dataset a Mϕ MGC gene signature was enriched in the squamous PDAC subtype and correlated with poorer overall survival (P = 0.018). Morphometric and immunofluorescence analyses further showed increased 53BP1+Ki67+ nuclei and nuclear atypia in MGCs, indicating ongoing proliferation despite DNA damage. Together, these data identify MGCs of Mϕ origin as an immune cell state shaped by hypoxia and stress signaling, associated with aggressive tumor phenotypes, and potentially exploitable as an immune classifier in PDAC.

Humans↗
PubMedDate not supplied

An interventional magnetic resonance imaging technique for the molecular characterization of intraprostatic dynamic contrast enhancement.

The biological characterization of an individual patient's tumor by noninvasive imaging will have an important role in cancer care and clinical research if the molecular processes that underlie the image data are known. Spatial heterogeneity in the dynamics of magnetic resonance imaging contrast enhancement (DCE-MRI) is hypothesized to reflect variations in tumor angiogenesis. Here we demonstrate the feasibility of precisely colocalizing DCE-MRI data with the genomic and proteomic profiles of underlying biopsy tissue using a novel MRI-guided biopsy technique in a patients with prostate cancer.

Biopsy↗
PubMed2026

Integrative quantum and systems biology of cancer: From molecular fluctuations to ecological outcomes.

This review treats cancer as a multiscale adaptive system, asks what the framework must predict to be worth adopting, and separates at each scale what the evidence establishes from what is proposed. It is an expert narrative synthesis, not a systematic review, and states the limits of that design. Proton transfer and tautomeric shifts contribute to spontaneous mispairing but do not license claims of directed or non-random mutation: replication timing, three-dimensional chromatin organization, sequence context and known mutagenic processes explain most mutational heterogeneity, leaving any quantum contribution as a residual against that baseline. The Waddington quasi-potential is bounded: outside detailed balance the dynamics are not gradient-derivable and require a probability-flux term. Hysteresis, rate-limited bimodality and return to state after perturbation distinguish an attractor from a transcriptomic cluster. Single-cell karyotype and live-imaging evidence supports whole-genome doubling as an unstable intermediate of heterogeneous origin and context-dependent consequence, not a uniform adaptive strategy. Systems and synthetic biology, virtual cells and digital twins are assessed against benchmarks, not promise. Tissue-scale ecology is reported with the spatial measurements now quantifying it, including evidence that stromal niche construction is not uniformly tumor-supporting. RNA modification is a layer in its own right, showing that the interpretation of a regulatory signal, not its magnitude, is biologically decisive. A dedicated section states the framework's commitments, the observable and evidence at each scale, and what would falsify them, asking what this adds to somatic mutation theory with clonal evolution and plasticity.

Neoplasms↗
PubMed2003

Regulatory gene networks and the properties of the developmental process.

Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.

Animals↗
PubMed1997

In vitro RNA binding of the hepatitis A virus proteinase 3C (HAV 3Cpro) to secondary structure elements within the 5' terminus of the HAV genome.

The secondary structure elements at the 5' nontranslated region (NTR) of the picornaviral RNAs can be divided functionally into two domains, one of which directs cap-independent translation, whereas the other is essential for viral RNA replication. For the latter, the formation of an RNA replication complex that involves particularly viral proteinase-containing polypeptides and cellular proteins has been shown (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598; Xiang W et al., 1995, RNA 1:892-904). To initiate studies on the formation of the hepatitis A virus (HAV) RNA replication complex, binding of the HAV proteinase 3Cpro and 3CD to secondary structure elements at the 5' and 3' NTR of the HAV RNA was investigated. Using mobility shift assay, UV crosslinking/ label transfer, and northwestern analysis, we show that both the HAV 3Cpro and the proteolytically inactive mutant bind to in vitro synthesized transcripts, suggesting that the RNA-binding site of the enzyme is separated spatially from its catalytic center. Weak interactions with HAV 3Cpro were found for individual secondary structure elements comprising less than 100 nt. RNA-binding specificity was unambiguous for transcripts comprising at least two stem-loops along with the polypyrimidine tract. Furthermore, competition experiments suggest that the 5' terminus of the HAV genome contains multiple binding sites for HAV 3Cpro. In contrast to poliovirus, binding capacity of HAV 3CD to RNA of the 5' NTR was not improved as compared to 3C. The data imply that, during the viral life cycle, HAV 3Cpro might serve replicative function(s) in addition to proteolysis of the viral polyprotein.

3C Viral Proteases↗
PubMed1992

cis and trans requirements for the selective packaging of adenovirus type 5 DNA.

Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome.

Adenoviruses, Human↗
PubMed1993

Six members of the mouse forkhead gene family are developmentally regulated.

The 110-aa forkhead domain defines a class of transcription factors that have been shown to be developmentally regulated in Drosophila melanogaster and Xenopus laevis. The forkhead domain is necessary and sufficient for target DNA binding as shown for the rat hepatic nuclear factor 3 (HNF3) gene family. We have cloned six forkhead gene family members from a mouse genomic library in addition to the mouse equivalents of the genes for HNF3 alpha, -beta, and -gamma. The six genes, termed fkh-1 to fkh-6, share a high degree of similarity with the Drosophila forkhead gene, having 57-67% amino acid identity within the forkhead domain. fkh-1 seems to be the mammalian homologue of the Drosophila FD1 gene, as the sequences are 86% identical. fkh-1 to fkh-6 show distinct spatial patterns of expression in adult tissues and are expressed during embryogenesis.

Amino Acid Sequence↗
PubMed2006

Global studies of cell type-specific gene expression in plants.

Technological advances in expression profiling and in the ability to collect minute quantities of tissues have come together to allow a growing number of global transcriptional studies at the cell level in plants. Microarray technology, with a choice of cDNA or oligo-based slides, is now well established, with commercial full-genome platforms for rice and Arabidopsis and extensive expressed sequence tag (EST)-based designs for many other species. Microdissection and cell sorting are two established methodologies that have been used in conjunction with microarrays to provide an early glimpse of the transcriptional landscape at the level of individual cell types. The results indicate that much of the transcriptome is compartmentalized. A minor but consistent percentage of transcripts appear to be unique to specific cell types. Functional analyses of cell-specific patterns of gene expression are providing important clues to cell-specific functions. The spatial dissection of the transcriptome has also yielded insights into the localized mediators of hormone inputs and promises to provide detail on cell-specific effects of microRNAs.

Expressed Sequence Tags↗
PubMed2006

Generation of multipurpose alleles for the functional analysis of the mouse genome.

A novel generation of retroviral gene-trap vectors has been developed with the ability to induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems, which can repair and re-induce the gene-trap mutations when activated in succession. After the gene-trap insertions are passaged into mice, this system enables the induction of temporally and spatially restricted mutations in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. These vectors have been used to assemble the largest library of ES cell lines with conditional mutations in single genes, presently totalling 1724 unique genes. Due to their efficiency, the vectors are part of the core technologies to be used by EUCOMM for establishing a complete collection of conditional null mutations in mice.

Alleles↗
PubMed1999

p53 expression and regulation by NMDA receptors in the developing rat brain.

The p53 tumor suppressor gene, which is considered the guardian of the genome, encodes a phosphoprotein, which is a sequence-specific transcriptional activator or repressor of target genes. The role of p53 in developmental processes has not been studied extensively, although its expression appears to undergo temporal and spatial changes during prenatal and postnatal development. In the present study, we assessed the levels of p53 mRNA and protein in the developing rat brain and its relation to developmental cell death. Furthermore, we investigated the potential role of n-methyl-d-aspartate (NMDA) receptors in regulating p53 expression, since these receptors are involved in the control of cell death. We found that p53 mRNA and protein were detectable in the rat brain throughout perinatal development. In embryos, p53 immunoreactivity was mainly localized in the nuclei of neuroepithelial cells, with a maximum in staining at embryonic day (E)12. In the neuroepithelium, we also found significant numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells, both in dividing periventricular cells and in migrating neurons. In neonates, immediately after birth there was a reduction in the number of apoptotic cells, which then increased to reach a maximum at postnatal day (P)5. Postnatally, apoptotic as well as p53-positive cells were detected in most brain areas. P53 immunoreactivity was also highest on P5. In most cells, p53 immunoreactivity and the TUNEL signal colocalized. P53 immunoreactivity as well as the number of TUNEL- positive cells were dramatically decreased in the brains of newborns treated with MK-801, an NMDA receptor antagonist. Our results show that p53 is involved in the control of developmental cell death, and that NMDA receptors play a regulatory role in the expression of the p53 gene, and thus in apoptosis occurring in the developing rat brain.

Animals↗
PubMed2002

M13 endopeptidases: New conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish.

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.

Amino Acid Motifs↗
PubMed2006

MUSTANG: a multiple structural alignment algorithm.

Multiple structural alignment is a fundamental problem in structural genomics. In this article, we define a reliable and robust algorithm, MUSTANG (MUltiple STructural AligNment AlGorithm), for the alignment of multiple protein structures. Given a set of protein structures, the program constructs a multiple alignment using the spatial information of the C(alpha) atoms in the set. Broadly based on the progressive pairwise heuristic, this algorithm gains accuracy through novel and effective refinement phases. MUSTANG reports the multiple sequence alignment and the corresponding superposition of structures. Alignments generated by MUSTANG are compared with several handcurated alignments in the literature as well as with the benchmark alignments of 1033 alignment families from the HOMSTRAD database. The performance of MUSTANG was compared with DALI at a pairwise level, and with other multiple structural alignment tools such as POSA, CE-MC, MALECON, and MultiProt. MUSTANG performs comparably to popular pairwise and multiple structural alignment tools for closely related proteins, and performs more reliably than other multiple structural alignment methods on hard data sets containing distantly related proteins or proteins that show conformational changes.

Algorithms↗
PubMed2004

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus.

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear beta-catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of beta-catenin is highly suggestive. We demonstrated that micro1 is a direct target of beta-catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.

Amino Acid Sequence↗
PubMed2004

Plant disease resistance protein signaling: NBS-LRR proteins and their partners.

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.

Gene Expression Regulation, Plant↗
PubMed2004

Gene expression in sheep skin and wool (hair).

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10(-4). In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin.

Animals↗
PubMedDate not supplied

Overlapping transcription units in the dopa decarboxylase region of Drosophila.

The many examples of overlap in the genes of various viruses and bacteria illustrate that the parsimonious utilization of the coding capacity of DNA is relatively common amongst prokaryotes. The recent discovery of a pupal cuticle gene within an intron of the completely unrelated Gart locus in Drosophila shows that overlapping transcription units also exist in higher organisms. However, the prevalence of such phenomena in unknown. We report here a quite different situation of overlap between the 3' termini of a pair of convergent transcription units in another region of the Drosophila genome. This 88-base-pair (bp) genomic region encodes the 3' terminus of the messenger RNA for the enzyme dopa decarboxylase (Ddc) and, in opposite orientation, the 3' terminus of the adjacent gene whose function is unknown. An analysis of the temporal and spatial distribution of the two transcripts within the organism shows that high levels of both transcripts are never concordant. However, within the testes, where the 3' transcript is maximally expressed, low levels of Ddc transcript were detected. This result raises the possibility that a hybrid molecule involving the two transcripts forms in vivo or that transcription interference occurs, with concomitant regulatory implications.

Animals↗