Multiple Profile Models Extract Features from Protein Sequence Data and Resolve Functional Diversity of Very Different Protein Families.

Functional classification of proteins from sequences alone has become a critical bottleneck in understanding the myriad of protein sequences that accumulate in our databases. The great diversity of homologous sequences hides, in many cases, a variety of functional activities that cannot be anticipated. Their identification appears critical for a fundamental understanding of the evolution of living organisms and for biotechnological applications. ProfileView is a sequence-based computational method, designed to functionally classify sets of homologous sequences. It relies on two main ideas: the use of multiple profile models whose construction explores evolutionary information in available databases, and a novel definition of a representation space in which to analyze sequences with multiple profile models combined together. ProfileView classifies protein families by enriching known functional groups with new sequences and discovering new groups and subgroups. We validate ProfileView on seven classes of widespread proteins involved in the interaction with nucleic acids, amino acids and small molecules, and in a large variety of functions and enzymatic reactions. ProfileView agrees with the large set of functional data collected for these proteins from the literature regarding the organization into functional subgroups and residues that characterize the functions. In addition, ProfileView resolves undefined functional classifications and extracts the molecular determinants underlying protein functional diversity, showing its potential to select sequences towards accurate experimental design and discovery of novel biological functions. On protein families with complex domain architecture, ProfileView functional classification reconciles domain combinations, unlike phylogenetic reconstruction. ProfileView proves to outperform the functional classification approach PANTHER, the two k-mer-based methods CUPP and eCAMI and a neural network approach based on Restricted Boltzmann Machines. It overcomes time complexity limitations of the latter.

Perception of environmental signals by a marine diatom.

Diatoms are a key component of marine ecosystems and are extremely important for the biogeochemical cycling of silica and as contributors to global fixed carbon. However, the answers to fundamental questions such as what diatoms can sense in their environment, how they respond to external signals, and what factors control their life strategies are largely unknown. We generated transgenic diatom cells containing the calcium-sensitive photoprotein aequorin to determine whether changes in calcium homeostasis are used to respond to relevant environmental stimuli. Our results reveal sensing systems for detecting and responding to fluid motion (shear stress), osmotic stress, and iron, a key nutrient that controls diatom abundance in the ocean.

Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms.

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.

Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products.

The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2-408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme delta 1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.

Transformation of Nonselectable Reporter Genes in Marine Diatoms.

: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.