Comparative and evolutionary analysis of α-amylase gene across monocots and dicots

α-amylase is an important enzyme involved in starch degradation to provide energy to the germinating seedling. The present study was conducted to reveal structural and functional evolution of this gene among higher plants. Discounting polyploidy, most plant species showed only a single copy of the gene making multiple isoforms in different tissues and developmental stages. Genomic length of the gene ranged from 1472 bp in wheat to 2369 bp in soybean, and the size variation was mainly due to differences in the number and size of introns. In spite of this variation, the intron phase distribution and insertion sites were mostly conserved. The predicted protein size ranged from 414 amino acid (aa) in soybean to 449aa in Brachypodium. Overall, the protein sequence similarity among orthologs ranged from 56.4 to 97.4 %. Key motifs and domains along with their relative distances were conserved among plants although several species, genera, and class specific motifs were identified. The glycosyl hydrolase superfamily domain length varied from 342aa in soybean to 384aa in maize and sorghum while length of the C-terminal β-sheet domain was highly conserved with 61aa in all monocots and Arabidopsis but was 59aa in soybean and Medicago. Compared to rice, 3D structure of the proteins showed 89.8 to 91.3 % similarity among the monocots and 72.7 to 75.8 % among the dicots. Sequence and relative location of the five key aa required for the ligand binding were highly conserved in all species except rice.     

4SALE – A tool for synchronous RNA sequence and secondary structure alignment and editing

Background  

In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database), we are faced with the problem to handle sequences and their associated secondary structures synchronously.     

Workflow to improve patient recruitment for clinical trials within hospital information systems – a case-study

Background

The identification of suitable patients is a common problem in clinical trials that is especially evident in tertiary care hospitals.

Methods

We developed and analysed a workflow, which uses routine data captured during patient care in a hospital information system (HIS), to identify potential trial subjects. Study nurses or physicians are notified automatically by email and verify eligibility.

Results

As a case study we implemented the system for acute myeloid leukemia (AML) trials in Münster. During a test period of 50 days 41 patients were identified by the system. 13 could be included as new trial patients, 7 were already included during earlier visits. According to review of paper records no AML trial patient was missed by the system. In addition, the hospital information system further allowed to preselect patients for specific trials based on their disease status and individual characteristics.

Conclusion

Routine HIS data can be used to support patient recruitment for clinical trials by means of an automated notification workflow.

     

Coproporphyrinogen III oxidase from barley and tobacco — sequence analysis and initial expression studies

Coproporphyrinogen III oxidase (coprogen oxidase; EC 1.3.3.3) is part of the pathway from 5-aminolevulinate to protoporphyrin IX which is common in all organisms and catalyses oxidative decarboxylation at two tetrapyrrole side chains. We cloned and sequenced fulllength cDNAs encoding coprogen oxidase from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum L.). They code for precursor peptides of 43.6 kDa and 44.9 kDa, respectively. Import into pea plastids resulted in a processed tobacco protein of approx. 39 kDa, which accumulated in the stroma fraction. Induction of synthesis of recombinant putative tobacco mature coprogen oxidase consisting of 338 amino-acid residues in Escherichia coli at 20°C result in a catalytically active protein of approx. 39 kDa, while induction of its formation at 37°C immediately terminated bacterial growth, possibly due to toxic effects on the metabolic balance of tetrapyrrole biosynthesis. The plant coprogen oxidase gene was expressed to different extents in all tissues investigated. This is most likely due to the differing requirements for tetrapyrroles in different organs. The steady-state level of mRNA did not significantly differ in etiolated and greening barley leaves. The content of coprogen oxidase RNA reached its maximum in developing cells and decreased drastically when cells were completely differentiated. Functioning of the two photosystems apparatus requires the synthesis of all pigment and protein components during plant development. It is speculated that the enzymes involved in tetrapyrrole synthesis are developmentally rather than light-dependently regulated. Regulation of these enzymes also guarantees a constant flux of metabolic intermediates and avoids photodynamic damage by accumulating porphyrins. Accession number: The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X82830 (barley coprogen oxidase) and X82831 (tobacco coprogen oxidase).     

DNA sequence analysis of a rat RT1 class 11 A β gene

The rat major histocompatibility complex (RT1) encodes twin sets of class II molecules, each consisting of two polypeptide chains referred to as A and A , and E and E . A gene encoding the RTLA chain was isolated from a rat genomic library using an HLA-DQ chain cDNA as a probe. The nucleotide sequence of the coding regions of this gene was determined. Comparison of this sequence with those of the corresponding genes of mouse (H-2A ) and human (HLA-DQ ) revealed that this gene has been highly conserved during evolution, and that some parts of the molecule are more conserved than others. Analysis of the nucleotide sequence encoding the two external domains suggests that the membrane proximal domain has been subject to conservative selection, whereas replacement substitutions have been selected positively at certain residues within the amino terminal domain. The overall organization of the RT1.A gene is similar to that of the H-2A gene.     

GeneNotes – A novel information management software for biologists

Background  

Collecting and managing information is a challenging task in a genome-wide profiling research project. Most databases and online computational tools require a direct human involvement. Information and computational results are presented in various multimedia formats (e.g., text, image, PDF, word files, etc.), many of which cannot be automatically processed by computers in biologically meaningful ways. In addition, the quality of computational results is far from perfect and requires nontrivial manual examination. The timely selection, integration and interpretation of heterogeneous biological information still heavily rely on the sensibility of biologists. Biologists often feel overwhelmed by the huge amount of and the great diversity of distributed heterogeneous biological information.     

Isolation and sequence analysis of a β-tubulin gene from arbuscular mycorrhizal fungi

A full-length β-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in β-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5′ and 3′ splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two β-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.     

Network genomics – A novel approach for the analysis of biological systems in the post-genomic era

Network Genomics studies genomics and proteomics foundations of cellular networks in biological systems. It complements systems biology in providing information on elements, their interaction and their functional interplay in cellular networks. The relationship between genomic and proteomic high-throughput technologies and computational methods are described, as well as several examples of specific network genomic application are presented.     

Polymorphism detection and sequence analysis of human T-cell receptor Vα-chain-encoding gene segments

The T-cell receptor (Tcr) provides specificity for antigen recognition by its variable domain, primarily consisting of two germline encoded variable (V) region gene segments. Thus it has been suggested that inherited polymorphisms in the TCRV gene segments could contribute to differential immune responsiveness (e.g., autoimmunity) in human populations. In the present study, we have sought potentially functional polymorphisms in the germline TCRAV gene segments. Using denaturing gradient gel electrophoresis on polymerase chain reaction (PCR)-amplified products from the pooled DNA of many individuals, we identified polymorphisms in the TCRAV2S1, AV4S1, AV7S1, and AV8S1 gene segments. A complete DNA sequence analysis of these PCR products identified polymorphisms that affected amino acids in the predicted antigen-binding regions of the Tcr chain, as well as polymorphisms in the introns. Genotype analysis of all nine DNA point mutations showed a 5%–50% range (averaging 35%) of minor allele frequencies, often resulting in individuals homozygous for the alternate allele forms. All possible haplotype combinations of the amino acid-affecting polymorphisms were found, indicating that in human populations there are a large number of different germline haplotypes encoding V gene segment alleles. These TCRAV coding region polymorphisms provide the rationale for, and allow the direct testing of, hypotheses concerning inherited polymorphisms within the T-cell receptor genes that may contribute to autoimmune susceptibility.The nucleotide sequence data reported in this paper have been submitted to the Genbank nucleotide sequence database and have been assigned the accession numbers L11159–L11162.     

METAPOP—A software for the management and analysis of subdivided populations in conservation programs

We introduce a computer program for the dynamic and flexible management of conserved subdivided populations. Using molecular marker data or pedigree information, the software determines the optimal contributions (i.e., number of offspring) of each individual, the number of migrants, and the particular subpopulations involved in the exchange of individuals in order to maintain the largest level of gene diversity in the whole population with a desired control in the rate of inbreeding. Restrictions can be introduced for the total number of migrants, and the mating of particular pairs and their contribution. A full genetic diversity analysis of the population is carried out. The optimal contribution from each subpopulation to a pool of maximal gene diversity is also provided by the program.     

Feeding studies take guts – critical review and recommendations of methods for stomach contents analysis in fish

Studies on the feeding ecology of fish are essential for exploring and contrasting trophic interactions and population and community dynamics within and among aquatic ecosystems. In this respect, many different methods have been adopted for the analysis of fish stomach contents. No consensus has, however, been reached for a standardised methodology despite that for several decades there has been an ongoing debate about which methodical approaches that should be preferred. Here, we critically review and scrutinise methods, addressing their strengths and weaknesses and emphasising inherent problems and possible pitfalls in their use. Although our critical assessment reveals that no completely ideal approach exists, appropriate and reliable procedures can be adopted through careful considerations and implementation. In particular, we advocate that different objectives require different methodical approaches and the choice of method should therefore be closely linked to the research questions that are addressed. For a standardisation of methods, we recommend a combination of the relative-fullness and presence–absence methods as the optimal approach for the commonly applied feeding studies addressing relative dietary composition in terms of prey diversity and abundance. Additionally, we recommend the gravimetric method for objectives related to the quantification of food consumption rates and the numerical method for prey selection studies. DNA-based dietary analysis provides a new and promising complementary approach to visual examination of stomach contents, although some technical challenges still exist. The suggested method standardisation facilitates comparisons across species, ecosystems and time and will enhance the applicability and benefits of fish feeding studies in trophic ecology research.     

Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

Laparoscopic Hepatectomy for Hepatic Colorectal Metastases – A Retrospective Comparative Cohort Analysis and Literature Review

Background

Laparoscopic hepatectomy (LH) for management of hepatic colorectal metastases (HCRM) is commonly being performed; however, there are limited reports comparing LH outcomes with those of open hepatectomy (OH) procedure. The aim of the present study was to compare perioperative outcomes between the LH and OH procedures performed at a single medical center.

Methods

From Jan 2008 to May 2012, 30 patients with pathologically confirmed HCRM underwent LH, and 140 patients underwent OH at our hospital. Patients'' demographics, perioperative outcomes were retrospectively analyzed.

Results

2 patients (6.7%) in the LH group underwent laparotomies for intraoperative hemorrhage. The LH group had an increased surgical duration (235 min vs. 365 min, (P<0.001), shorter hospital stay (7.5 days vs. 11.5 days, P<0.001), and fewer complications (26.2% vs. 55%, P<0.001) than the OH group. However, in a matched cohort comparison of 30 LH cases and 30 OH cases, no significant variations were observed in the following parameters: surgical duration (235 min vs. 255 min, P = 0.23), positive margin rates (6.7% vs. 0.0%, P = 0.27), or postoperative hematological changes. LH patients had less estimated blood loss (215 ml vs. 385 ml, P<0.001), less morbidity (26.2% vs. 50%, P = 0.02), shorter hospital stay (7.5 days vs. 11.5 days, P<0.001), and lower analgesic requests than with those in the OH group.

Conclusions

LH for metastatic colorectal cancer is a safe and feasible treatment, even in patients who underwent prior laparotomy surgeries and provides significantly less morbidity and shorter hospital stay than OH, without compromising curability or increasing morbidity.     

Decolourization of Industrial Effluents – Available Methods and Emerging Technologies – A Review

Water pollution control is presently one of the major thrust areas of scientific research. While coloured organic compounds generally impart only a minor fraction of the organic load to wastewaters, their colour renders them aesthetically unacceptable. Stringent regulating measures are coaxing industries to treat their waste effluents to increasingly high standards. Colour removal, in particular, has recently become an area of major scientific interest as indicated by the multitude of related research reports. During the past two decades, several decolourization techniques have been reported, few of which have been accepted by some industries. There is a need to find alternative treatments that are effective in removing dyes and colourants from large volume of effluents, which are cost-effective, like the biological or integrated systems. This article reviews some of the widely used and most promising industrial wastewater decolourization methods. Data on decolourizing efficiencies of different causative agents, obtained by means of different physical, chemical and biological methods are discussed. Further a critical review is made on the various treatment methodologies and emerging technologies with a note on their advantages and disadvantages.     

Laboratory information management system for membrane protein structure initiative – from gene to crystal

Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.     

CGHPRO – A comprehensive data analysis tool for array CGH

Background  

Array CGH (Comparative Genomic Hybridisation) is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios.     

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.     

A novel carotenoid biosynthesis gene coding for ζ-carotene desaturase: functional expression,sequence and phylogenetic origin

A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting -carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56 740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of -carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between -carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the -carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.