Optimizing the search algorithm for protein engineering by directed evolution.

An in silico protein model based on the Kauffman NK-landscape, where N is the number of variable positions in a protein and K is the degree of coupling between variable positions, was used to compare alternative search strategies for directed evolution. A simple genetic algorithm (GA) was used to model the performance of a standard DNA shuffling protocol. The search effectiveness of the GA was compared to that of a statistical approach called the protein sequence activity relationship (ProSAR) algorithm, which consists of two steps: model building and library design.

Putting engineering back into protein engineering: bioinformatic approaches to catalyst design.

Complex multivariate engineering problems are commonplace and not unique to protein engineering. Mathematical and data-mining tools developed in other fields of engineering have now been applied to analyze sequence-activity relationships of peptides and proteins and to assist in the design of proteins and peptides with specified properties. Decreasing costs of DNA sequencing in conjunction with methods to quickly synthesize statistically representative sets of proteins allow modern heuristic statistics to be applied to protein engineering.

TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution.

In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported for Srb8 and Srb9. Here, we identify a TRAP240/ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11.

Systematic variation of amino acid substitutions for stringent assessment of pairwise covariation.

During protein evolution, amino acids change due to a combination of functional constraints and genetic drift. Proteins frequently contain pairs of amino acids that appear to change together (covariation). Analysis of covariation from naturally occurring sets of orthologs cannot distinguish between residue pairs retained by functional requirements of the protein and those pairs existing due to changes along a common evolutionary path.

Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis.

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1.

Characteristics of hydroxypropyl methylcellulose influencing compactibility and prediction of particle and tablet properties by infrared spectroscopy.

Particle characteristics, chemical substitution, compaction behavior, and tablet properties of hydroxypropyl methylcellulose powders from two different suppliers were related using multivariate data analysis. By Principal Component Analysis it was shown that the the degree of substitution of the HPMC powders did not correlate to the particle and compaction properties as strongly as anticipated. Particle shape and powder surface area seem to be more important for the compaction behaviour of the powders than the degree of substitution.

Characterization of the mouse genes for mitochondrial transcription factors B1 and B2.

We have recently fully reconstituted the basal human mitochondrial transcription machinery in a pure in vitro system. Surprisingly, we found two different transcription factors (TFB1M and TFB2M) that each interact with mitochondrial RNA polymerase in human mitochondria, whereas there is only one such factor in budding yeast mitochondria. This unexpected finding raised important questions concerning the regulation of mitochondrial transcription in mammals in general and in other metazoans.

Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A.

Psychometric evaluation of a Swedish version of the Reiss Screen for Maladaptive Behavior.

Background: People with intellectual disability (ID) are afflicted by mental health problems to a greater extent than other individuals, and the coexistence of ID and mental health problems involves diagnostic as well as treatment difficulties.

Methods: A Swedish version of the Reiss Screen for Maladaptive Behavior (RSMB), an instrument used for identification of mental health problems in people with intellectual disability (ID) was evaluated in terms of inter-rater agreement, internal consistency, item grouping and criterion validity based on a random sample and a clinical group of adults with ID.

Results: The Swedish version of the RSMB had moderate-to-low inter-rater agreement on specific items and good internal consistency.

Reliability and responsiveness of three different pain assessments.

The visual analogue scale (VAS) and ordered categorical scales, i.e. numeric rating scales (NRS), are commonly used in the assessment of pain.

Exploration of sequence space for protein engineering.

The process of protein engineering is currently evolving towards a heuristic understanding of the sequence-function relationship. Improved DNA sequencing capacity, efficient protein function characterization and improved quality of data points in conjunction with well-established statistical tools from other industries are changing the protein engineering field. Algorithms capturing the heuristic sequence-function relationships will have a drastic impact on the field of protein engineering.

Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells.

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes.

Mediator--a universal complex in transcriptional regulation.

The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II-dependent genes in the Saccharomyces cerevisiae genome. Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter. It is now clear that Mediator-like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation.

Increased in vivo apoptosis in cells lacking mitochondrial DNA gene expression.

We have attempted to determine whether loss of mtDNA and respiratory chain function result in apoptosis in vivo. Apoptosis was studied in embryos with homozygous disruption of the mitochondrial transcription factor A gene (Tfam) and tissue-specific Tfam knockout animals with severe respiratory chain deficiency in the heart. We found massive apoptosis in Tfam knockout embryos at embryonic day (E) 9.

Structural organization of yeast and mammalian mediator complexes.

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II.

Directed evolution: the 'rational' basis for 'irrational' design.

The development of powerful genetic manipulation formats has revolutionized the creation of functional biological molecules. Recent advances in directed evolution demonstrate that multiple properties of proteins can be optimized simultaneously and rapidly. Improved proteins often contain multiple and dispersed substitutions that act synergistically to improve enzyme properties and function.

Mediator-nucleosome interaction.

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size.

Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.

A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2.

Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe.

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S.

Characterisation of particle properties and compaction behaviour of hydroxypropyl methylcellulose with different degrees of methoxy/hydroxypropyl substitution.

The particle characteristics and compaction behaviour of hydroxypropyl methylcellulose (HPMC) powders from two different suppliers were studied regarding effects of methoxy/hydroxypropyl substitution. Samples included Methocel K4M (low substitution ratio), E4M (medium) and F4M (high) and the corresponding substitution ratios from Metolose: 90 SH 4000, 60 SH 4000, and 65 SH 4000. Characterisation of the particle properties and compaction behaviour of the pure polymers suggested that reported differences in drug release behaviour of Methocel E4M compared with the other two powders may be related to the lower powder surface area, differing particle morphology and lower fragmentation propensity during compaction.

Use of tablet tensile strength adjusted for surface area and mean interparticulate distance to evaluate dominating bonding mechanisms.

In this study, tablet tensile strength has been adjusted for tablet surface area and the average distance between particles in compacts of different materials. The aim of the study was to evaluate the feasibility of using this concept to assess the dominating interparticulate bonding mechanisms. Adjustment of the tensile strength for both tablet surface area and mean pore radius gave similar bonding strength values for materials bonding mainly by weak distance forces (crystalline lactose, sucrose, and microcrystalline cellulose) almost independently of compaction pressure.

Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli.

The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res.

Cellulose powder from Cladophora sp. algae.

The surface are and crystallinity was measured on a cellulose powder made from Cladophora sp. algae. The algae cellulose powder was found to have a very high surface area (63.