Damage-induced chromatome dynamics link Ubiquitin ligase and proteasome recruitment to histone loss and efficient DNA repair

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Mutational analysis of the "regulatory module" of hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.     

Automated microsequence analysis in the presence of a synthetic "carrier"

When small amounts of protein are subjected to automated sequence analysis, significant material washes out during the solvent wash steps and prevents extended analysis. Inclusion of a synthetic “carrier,” succinylated poly-ornithine, with the protein to be sequenced significantly reduces protein washout and permits extended automated microsequence analysis. This carrier also permits microquantities of protein to be sequenced in the presence of the detergent, sodium dodecyl sulfate.     

Spatial structures in a reaction-diffusion system--detailed analysis of the "Brusselator"

Continuous dependence of spatially nonuniform concentration profiles for the 'Brussellator" reaction mechanims on the characteristic length of the system is given both for zero flux and fixed boundary conditions. Branches of solutions arising through primary bifurcation form closed curves. Secondary bifurcations giving rise to spatially asymmetric solutions exist for fixed boundary conditions. Results of a stability analysis of individual solutions are discussed. A method of composing complex spatial profiles for higher lengths from elementary solutions for smaller lenghts is suggested and tested in the case of zero flux boundary conditions. Emergence of subsequently more complex stable patterns in dependence on increasing length of the system suggests many similarities to gradual build up of complex morphogenetic patterns.     

An analysis of the meaning of the concepts of "commitment" and "determination" in the study of patterns of development

The use of the term "commitment" by different authors was compared and the term itself was compared with the term "determination". Different authors understand the term "commitment" in different ways. It is proposed to preserve the terms "competence", "determination" (labile and stable) and "differentiation" in studies of normal development at stages preceding the appearance of organ rudiments in order to facilitate the use of the knowledge acquired by experimental embryology and to decipher these concepts at the molecular level. The meaning of the term "commitment" should be made more precise when describing the experimental results and also when assessing the results obtained by various authors and published in numerous papers and reviews.     

Proteomic analysis of mouse oocytes reveals 28 candidate factors of the "reprogrammome"

The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date, limitations on oocyte numbers and proteomic technology have impeded this task, and the search for reprogramming factors has been conducted in embryonic stem (ES) cells instead. Here, we present the proteome of metaphase II mouse oocytes to a depth of 3699 proteins, which substantially extends the number of proteins identified until now in mouse oocytes and is comparable by size to the proteome of undifferentiated mouse ES cells. Twenty-eight oocyte proteins, also detected in ES cells, match the criteria of our multilevel approach to screen for reprogramming factors, namely nuclear localization, chromatin modification, and catalytic activity. Our oocyte proteome catalog thus advances the definition of the "reprogrammome", the set of molecules--proteins, RNAs, lipids, and small molecules--that enable reprogramming.     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.     

A partial phylogenetic analysis of the "flavobacter-bacteroides" phylum: basis for taxonomic restructuring

On the basis of small subunit rRNA sequence analyses five major subgroups within the flavobacteria-bacteroides phylum have been defined. These are tentatively designated the cytophaga subgroup (comprising largely Cytophaga species), the flavobacter subgroup (comprising the true flavobacteria and the polyphyletic genus Weeksella), the bacteroides subgroup (comprising the bacteroides and certain cytophaga-like bacteria), the sphingobacter subgroup (which contains the known sphingolipid-producing members of the phylum), and the saprospira subgroup (comprising particular species of Flexibacter, Flavobacterium, Haliscomenobacter, and, of course, the genus Saprospira). These groupings are given not only by evolutionary distance analysis, but can be defined and distinguished on the basis of a simple small subunit rRNA signatures.     

Mutational analysis of the "turn" of helix clamp motif of HIV-1 reverse transcriptase

Helix clamp motifs of polymerases possessing the helix-turn-helix secondary structure are crucial for their polymerase activity by binding to the nucleic acid template/primer via the alpha helices. To study the functions of turn in helix clamp motif of human immunodeficiency virus (HIV)-1 RT, clones with turn mutants at rt271-274 of HIV-1 RT were generated and studied by one cycle infection assay. Mutants rtY271A and rtI274A almost lost their replication competency, while mutants rtA272P, rtA272S, and rtG273A retained comparable replication competency relative to wild type pseudotyped HIV-1. To study the mechanisms involved, RT proteins from rt271 to rt274 mutants were expressed and assayed for their RNA dependent DNA polymerase activity, DNA binding activity and processivity. Discordance between RT activity and viral replication efficiency of some turn mutants was observed, indicating that aside from RT, other steps in HIV replication could be affected by substitutions at the turn of helix clamp motif.     

Global analysis of a "simple" proteome: Methanococcus jannaschii

The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein-protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.     

A molecular phylogenetic analysis of the "true thrushes" (Aves: Turdinae)

The true thrushes (Passeriformes: Muscicapidae, subfamily Turdinae) are a speciose and widespread avian lineage presumed to be of Old World origin. Phylogenetic relationships within this assemblage were investigated using mitochondrial DNA (mtDNA) sequence data that included the cytochrome b and ND2 genes. Our ingroup sampling included 54 species representing 17 of 20 putative turdine genera. Phylogenetic trees derived via maximum parsimony and maximum likelihood were largely congruent. Most of the Turdine taxa sampled can be placed into one of six well supported clades. Our data indicate a polyphyletic Zoothera which can be divided into at least two (Afro-Asian and Austral-Asian) main clades. The genus Turdus, as presently recognized, is paraphyletic but forms a well supported clade with the addition of three mostly monotypic genera (Platycichla, Nesocichla, and Cichlherminia). We identify an exclusively New World clade that includes a monophyletic Catharus, Hylocichla, Cichlopsis, Entomodestes, Ridgwayia, and Ixoreus. Members of the morphologically and behaviorally distinct genera Sialia, Myadestes, and Neocossyphus unexpectedly form a basal clade. Using multiple outgroup choices, we show that this group is distantly related, but unequivocally the sister group to the remaining Turdines sampled. The Turdinae appear to be a relatively old songbird lineage, originating in the mid to late Miocene. If the Turdinae are indeed Old World in origin, our data indicate a minimum of three separate invasions of the New World.     

Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.