Localizing Genes to Cerebellar Layers by Classifying ISH Images

Gene expression controls how the brain develops and functions. Understanding control processes in the brain is particularly hard since they involve numerous types of neurons and glia, and very little is known about which genes are expressed in which cells and brain layers. Here we describe an approach to detect genes whose expression is primarily localized to a specific brain layer and apply it to the mouse cerebellum. We learn typical spatial patterns of expression from a few markers that are known to be localized to specific layers, and use these patterns to predict localization for new genes. We analyze images of in-situ hybridization (ISH) experiments, which we represent using histograms of local binary patterns (LBP) and train image classifiers and gene classifiers for four layers of the cerebellum: the Purkinje, granular, molecular and white matter layer. On held-out data, the layer classifiers achieve accuracy above 94% (AUC) by representing each image at multiple scales and by combining multiple image scores into a single gene-level decision. When applied to the full mouse genome, the classifiers predict specific layer localization for hundreds of new genes in the Purkinje and granular layers. Many genes localized to the Purkinje layer are likely to be expressed in astrocytes, and many others are involved in lipid metabolism, possibly due to the unusual size of Purkinje cells.     

Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.     

Comparative analysis of colorectal carcinoma cell lines that differ in metastatic potential

The present work is an attempt to compare cancer cells of the same origin but differing in the expression of CEA protein, a clinical marker of metastatic carcinomas. CEA, presumably, is one of the key factors in metastatic activity. We investigated the morphology of cell colonies in vitro, the expression pattern of epithelial markers, and the ability of these cells to form tumors and metastases in vivo. Their stem component was evaluated with a suicidal genetic construct sensitive to Oct4, an embryonic stem cell marker.     

In vitro derivation and characterization of a colorectal cancer stem cell subpopulation

In the present publication we describe for the first time the derivation of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells’ primary role in the development of malignant neoplasms.     

DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif

The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.     

Novel heterophile chicken antigen: immunohistochemical localization using antisera to Mycobacterium smegmatis and possible association with lymphocyte maturation

Summary A novel heterophile antigen shared byMycobacterium smegmatis and chicken tissues was demonstrated by the indirect immunoperoxidase method using antisera raised in rabbits immunized with a complete Freund's adjuvant containing killedMycobacterium smegmatis as an immunostimulating component. This antigen was strongly expressed in medullary lymphocytes of the thymus and bursa of Fabricius, but was undetectable in lymphoid cells of the cortical regions of these organs. Only a few lymphocytes stained positively for the antigen in T- and B-cell areas of the spleen. These data suggest that the heterophile antigen is associated with the intrathymic and intrabursal maturation of chicken lymphocytes. The antigen was also detected in some nonlymphoid cells. It was not found in sheep erythrocytes, human and rat tissues or in killed bacillus Culmette—Guerin.     

Lampbrush chromosomes in the japanese quail Coturnix coturnix japonica: cytological maps of macro chromosomes and meiotic crossover frequency in females

Cytological maps of lampbrush macrobivalents of the Japanese quail (Coturnix coturnix japonica) were constructed. Investigation of chiasmata allowed determination of the meiotic frequency of reciprocal genetic recombination (crossing over) in Japanese quail females. The total chiasma number in bivalents of Japanese quail oocyte nuclei was determined to be 53-58. Macrobivalents 1-5 and Z of the Japanese quail had on average 3.3 chiasmata per bivalent, and microbivalents, 1.0-1.1 chiasmata per bivalent. The chiasmata (crossover) frequency in Japanese quail females was lower than in chicks. In macrochromosomes of Japanese quail females, one crossover occurred per 43.9 Mb, and in chicken, per 30.0 Mb. Judging from chiasma frequency, the genetic length of the Japanese quail genome is likely to be 2650-2900 cM. Crossover frequency in the species was 0.023 per Mb in macrobivalents and 0.07-0.08 Mb in microbivalents and for the total genome, 0.041 crossovers per Mb. The genetic length of one Mb (theta) in female Japanese quails was 1.14 cM in macrochromosomes, 3.60-4.12 cM in microchromosomes, and about 1.96-2.15 cM averaged over the genome.