Sequence of three members and expression of a new major subfamily of glutelin genes from rice

Three members have been isolated of an additional glutelin gene subfamily, named subfamily B, consisting of about five members per haploid rice genome. Restriction fragment length polymorphism analysis showed major differences between Japonica and Indica lines, indicating the divergence of the subfamily since the split between the two varieties. While corresponding exons of the subfamily B showed 80 to 88% nucleotide sequence homology, those exons were only 60–65% homologous to those of the glutelin A subfamily [15, 19, 24], distinguishing them from the subfamily A. Intron position and derived polypeptide structure, in addition to the nucleotide sequence, confirm the subfamily B members as glutelins. Analysis of RNA from seeds of different stages of development showed that the subfamily B members were expressed at the same time as those of subfamily A, demonstrating coordinated regulation of the two subfamilies.     

Development of a hemolytic plaque assay for glutamic acid, lysine-containing polypeptides: demonstration that nonresponder mice produce antibodies to these peptides when conjugated to an immunogenic carrier.

Fowl gamma-globulin, when chemically conjugated to GLO or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLO and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLO responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLO responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.     

dChipSNP: significance curve and clustering of SNP-array-based loss-of-heterozygosity data

MOTIVATION: Oligonucleotide microarrays allow genotyping of thousands of single-nucleotide polymorphisms (SNPs) in parallel. Recently, this technology has been applied to loss-of-heterozygosity (LOH) analysis of paired normal and tumor samples. However, methods and software for analyzing such data are not fully developed. RESULT: Here, we report automated methods for pooling SNP array replicates to make LOH calls, visualizing SNP and LOH data along chromosomes in the context of genes and cytobands, making statistical inference to identify shared LOH regions, clustering samples based on LOH profiles and correlating the clustering results to clinical variables. Application of these methods to prostate and breast cancer datasets generates biologically important results. AVAILABILITY: The software module dChipSNP implementing these methods is available at http://biosun1.harvard.edu/complab/dchip/snp/ SUPPLEMENTARY INFORMATION: The breast cancer data are provided by Andrea L. Richardson, Zhigang C. Wang and James D. Iglehart.     

Brief inactivation of c-Myc is not sufficient for sustained regression of c-Myc-induced tumours of pancreatic islets and skin epidermis

Background  

Tumour regression observed in many conditional mouse models following oncogene inactivation provides the impetus to develop, and a platform to preclinically evaluate, novel therapeutics to inactivate specific oncogenes. Inactivating single oncogenes, such as c-Myc, can reverse even advanced tumours. Intriguingly, transient c-Myc inactivation proved sufficient for sustained osteosarcoma regression; the resulting osteocyte differentiation potentially explaining loss of c-Myc's oncogenic properties. But would this apply to other tumours?     

Measuring kinesin's first step

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.     

Structural and functional imaging with carbon nanotube AFM probes

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.     

Mapping parathyroid hormone, β-globin,insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes

Summary Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, -globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, -globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.