Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins.

We show, using dot matrix comparisons and statistical analysis of sequence alignments, that seven sequenced sigma factors, E. coli sigma-70 and sigma-32, B. subtilis sigma-43 and sigma-29, phage SP01 gene products 28 and 34, and phage T4 gene product 55, comprise a homologous family of proteins. Sigma-70, sigma-32, and sigma-43 each have two copies of a sequence similar to the helix-turn-helix DNA binding motif seen in CRP, and lambda repressor and cro proteins. B. subtilis sigma-29, SP01 gp28, and SP01 gp34 have at least one copy similar to this sequence. We propose that a second sequence, conserved in all seven proteins is the core RNA polymerase binding site. A third region, present only in sigma-70 and sigma-43, may also be involved in interaction with core. Available mutational evidence supports our model for sigma factor structure.     

Crystallization and preliminary x-ray diffraction studies of recombinant rabbit interferon-gamma

Two different crystal forms of recombinant rabbit IFN-gamma were obtained under different crystallization conditions. The first, a tetragonal form with space group P43212 or P41212, was obtained through vapor phase equilibration using the sitting drop rods technique with ammonium citrate as the major precipitating agent. The unit cell dimensions of this crystal form are a = b = 82.1 A and C = 116.3 A. These crystals diffract to 2.8 A resolution and contain a dimer in the asymmetric unit. A second crystal form was obtained by the batch method at pH 8.0 using sodium chloride as the precipitating agent. The crystals are hexagonal, space group P6122 or P6522, and with unit cell dimensions of a = b = 58.0 A and c = 169 A. This form contains monomer in the asymmetric unit and diffracts to greater than 2.7 A resolution. Both forms appear to be eminently suitable for further analyses and crystal structure solution.     

An integrated Arabidopsis annotation database for Affymetrix Genechip data analysis, and tools for regulatory motif searches

Genome-scale sequencing projects have provided the essential information required for the construction of entire genome chips or microarrays for RNA expression studies. The Arabidopsis and rice genomes have been sequenced and whole-genome oligonucleotide arrays are being manufactured. These should soon become available to researchers. Expression studies using genomic-scale expression arrays are providing us with a vast quantity of information at a rapid pace. The rate-limiting step in this type of experiments is not the data generation step but rather the data analysis component of experiments. We report improvements that should facilitate the analysis of Affymetrix Genechip expression data.     

Arabidopsis proteins containing similarity to the universal stress protein domain of bacteria

We have collected a set of 44 Arabidopsis proteins with similarity to the USPA (universal stress protein A of Escherichia coli) domain of bacteria. The USPA domain is found either in small proteins, or it makes up the N-terminal portion of a larger protein, usually a protein kinase. Phylogenetic tree analysis based upon a multiple sequence alignment of the USPA domains shows that these domains of protein kinases 1.3.1 and 1.3.2 form distinct groups, as do the protein kinases 1.4.1. This indicates that their USPA domain structures have diverged appreciably and suggests that they may subserve distinct cellular functions. Two USPA fold classes have been proposed: one based on Methanococcus jannaschii MJ0577 (1MJH) that binds ATP, and the other based on the Haemophilus influenzae universal stress protein (1JMV), highly similar to E. coli UspA, which does not bind ATP. A set of common residues involved in ATP binding in 1MJH and conserved in similar bacterial sequences is also found in a distinct cluster of Arabidopsis sequences. Threading analysis, which examines aspects of secondary and tertiary structure, confirms this Arabidopsis sequence cluster as highly similar to 1MJH. This structural approach can distinguish between the characteristic fold differences of 1MJH-like and 1JMV-like bacterial proteins and was used to assign the complete set of candidate Arabidopsis proteins to one of these fold classes. It is clear that all the plant sequences have arisen from a 1MJH-like ancestor.     

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.     

PEPPLOT, a protein secondary structure analysis program for the UWGCG sequence analysis software package.

We describe a program for the analysis of protein secondary structure that operates with the Sequence Analysis Software Package of the University of Wisconsin Genetics Computer Group (UWGCG). The program produces both graphic and printed output. Structure prediction using the Chou and Fasman and Robson et al methods, and hydropathy analysis by the method of Kyte and Doolittle are included along with a simplified method of hydrophobic moment analysis. The power of the program is the coordinated presentation of many different kinds of structural information on the same plot.     

Estimating and evaluating the statistics of gapped local-alignment scores.

We present a novel maximum-likelihood-based algorithm for estimating the distribution of alignment scores from the scores of unrelated sequences in a database search. Using a new method for measuring the accuracy of p-values, we show that our maximum-likelihood-based algorithm is more accurate than existing regression-based and lookup table methods. We explore a more sophisticated way of modeling and estimating the score distributions (using a two-component mixture model and expectation maximization), but conclude that this does not improve significantly over simply ignoring scores with small E-values during estimation. Finally, we measure the classification accuracy of p-values estimated in different ways and observe that inaccurate p-values can, somewhat paradoxically, lead to higher classification accuracy. We explain this paradox and argue that statistical accuracy, not classification accuracy, should be the primary criterion in comparisons of similarity search methods that return p-values that adjust for target sequence length.     

Spontaneous symmetry breaking in genome evolution

The quest for evolutionary mechanisms providing separation between the coding (exons) and noncoding (introns) parts of genomic DNA remains an important focus of genetics. This work combines an analysis of the most recent achievements of genomics and fundamental concepts of random processes to provide a novel point of view on genome evolution. Exon sizes in sequenced genomes show a lognormal distribution typical of a random Kolmogoroff fractioning process. This implies that the process of intron incretion may be independent of exon size, and therefore could be dependent on intron-exon boundaries. All genomes examined have two distinctive classes of exons, each with different evolutionary histories. In the framework proposed in this article, these two classes of exons can be derived from a hypothetical ancestral genome by (spontaneous) symmetry breaking. We note that one of these exon classes comprises mostly alternatively spliced exons.