In Arabidopsis thaliana the putative mitochondrial RNA helicases PMH1 and PMH2 are members of the large DEAD-box protein family. Our previous characterization of these proteins revealed that PMH1 and/or PMH2 are part of high molecular weight complexes. Now T-DNA insertion lines were established and characterized for each of these genes. Immunodetection analysis of cell suspension cultures established from pmh1-1 and pmh2-1 mutants revealed that indeed both DEAD-box proteins are detectable in large protein complexes with PMH2 being much more abundant than PMH1. In plants the knockout of PMH2 leads to reduced group II intron splicing efficiency. In addition the steady-state levels of several mature mitochondrial mRNAs are decreased while transcription is not influenced. This molecular phenotype suggests that PMH2 acts at the posttranscriptional level with a potential function as RNA chaperone required for formation or maintenance of complex RNA secondary structures of introns rather than a direct role in splicing. In contrast, the investigation of a pmh1-1 knockout line did not reveal any influence of this protein on processing and abundance of mitochondrial transcripts.

This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements.

Posttranscriptional processes are important for regulation of gene expression in plant mitochondria. DEAD-box proteins, which form a huge protein family with members from all kingdoms, are fundamental components in virtually all types of processes in RNA metabolism. Two members of this protein family, designated PMH1 and PMH2 (for PUTATIVE MITOCHONDRIAL RNA HELICASE), were analyzed and characterized in mitochondria of Arabidopsis (Arabidopsis thaliana). Green fluorescent protein tagging with N-terminal PMH1 and PMH2 sequences supports the mitochondrial localization of these proteins. Northern experiments, as well as histochemical b-glucuronidase staining of transgenic plants carrying respective promoter:b-glucuronidase fusion constructs, revealed differing transcription patterns for the two genes. In response to cold, however, transcript levels of both genes increased. Immunodetection analyses of mitochondrial protein complexes after two-dimensional blue native/urea SDS-PAGE and after fractionation on sucrose gradients strongly suggest that one or both proteins are part of RNA-dependent complexes. Cold treatment of cell cultures or solubilization of mitochondria in the presence of MgCl 2 favored the detection of high-molecular-mass complexes. This study paves the way for detailed analysis of high-molecular-mass complexes in mitochondria of higher plants.

We present an intensity-based nonrigid registration approach for the normalization of 3-D multichannel microscopy images of cell nuclei. A main problem with cell nuclei images is that the intensity structure of different nuclei differs very much; thus, an intensity-based registration scheme cannot be used directly. Instead, we first perform a segmentation of the images from the cell nucleus channel, smooth the resulting images by a Gaussian filter, and then apply an intensity-based registration algorithm. The obtained transformation is applied to the images from the nucleus channel as well as to the images from the other channels. To improve the convergence rate of the algorithm, we propose an adaptive step length optimization scheme and also employ a multiresolution scheme. Our approach has been successfully applied using 2-D cell-like synthetic images, 3-D phantom images as well as 3-D multichannel microscopy images representing different chromosome territories and gene regions. We also describe an extension of our approach, which is applied for the registration of 3D + t (4-D) image series of moving cell nuclei.

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