Structure of Escherichia coli dGTP Triphosphohydrolase: A HEXAMERIC ENZYME WITH DNA EFFECTOR MOLECULES*

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present study, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (K_d ∼ 50 nm). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K_m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.     

Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells

We describe a female patient with developmental delay, dysmorphic features and multiple congenital anomalies who presented a normal G-banded karyotype at the 550-band resolution. Array and multiplex-ligation probe amplification (MLPA) techniques identified an unexpected large unbalanced genomic aberration: a 17.6Mb deletion of 9p associated to a 14.8 Mb duplication of 20p. The deleted 9p genes, especially CER1 and FREM1, seem to be more relevant to the phenotype than the duplicated 20p genes. This study also shows the relevance of using molecular techniques to make an accurate diagnosis in patients with dysmorphic features and multiple anomalies suggestive of chromosome aberration, even if on G-banding their karyotype appears to be normal. Fluorescence in situ hybridization (FISH) was necessary to identify a masked balanced translocation in the patient's mother, indicating the importance of associating cytogenetic and molecular techniques in clinical genetics, given the implications for patient management and genetic counseling.     

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.     

Calculating phenotypic similarity between genes using hierarchical structure data based on semantic similarity

Phenotypic similarity is correlated with a number of measures of gene function, such as relatedness at the level of direct protein-protein interaction. The phenotypic effect of a deleted or mutated gene, which is one part of gene annotation, has caught broad attention. However, there have been few measures to study phenotypic similarity with the data from Human Phenotype Ontology (HPO) database, therefore more analogous measures should be developed and investigated. We used five semantic similarity-based measures (Jiang and Conrath, Lin, Schlicker, Yu and Wu) to calculate the human phenotypic similarity between genes (PSG) with data from HPO database, and evaluated their accuracy with information of protein-protein interaction, protein complex, protein family, gene function or DNA sequence. Compared with the gene pairs that were random selected, the results of these methods were statistically significant (all P<0.001). Furthermore, we assessed the performance of these five measures by receiver operating characteristic (ROC) curve analysis, and found that most of them performed better than the previous methods. This work had proved that these measures based on semantic similarity for calculation of PSG were effective for hierarchical structure data. Our study contributes to the development and optimization of novel algorithms of PSG calculation and provides more alternative methods to researchers as well as tools and directions for PSG study.     

Systematic classification of melanoma cells by phenotype-specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Carbon source utilization by the marine Dendryphiella species D. arenaria and D. salina

Carbon utilization by the marine Dendryphiella species, D. arenaria and D. salina, was investigated to detect differences in utilization and traits associated with their adaptation to the marine habitat. Fifty-four strains were isolated world-wide and tested for the utilization of various carbon sources using BIOLOG phenotype MicroArray (PM) and for the production of extracellular enzymes on solid culture media and on API ZYM assay strips. PM analysis showed that the fastest growth occurred on several monosaccharides and amino acids, 2-keto-d-gluconic acid, succinamide and turanose. Some polyols were poor carbon sources. However, the two species differed in their utilization rates of carbon sources, forming three major clusters: two separate clusters for D. arenaria and D. salina and a third cluster in which strains of the two species formed separate subclades that correlated with geographic origin. Several carbon sources were also found useful in differentiating the two speices. Dendryphiella salina did not utilize xylitol and quinic acid, whereas D. arenaria grew well on these substrates. The latter failed to grow on sorbitol and grew slowly on mannitol, both were good substrates for the former. There were also no qualitative differences between the extracellular enzymes produced, although laccase and peroxidase activities were confined only to some strains. The physiological similarities exhibited by the two species support the close relationship between D. arenaria and D. salina.     

Analysis of human disease genes in the context of gene essentiality

The characteristics of human disease genes were investigated through a comparative analysis with mouse mutant phenotype data. Mouse orthologs with mutations that resulted in discernible phenotypes were separated from mutations with no phenotypic defect, listing ‘phenotype’ and ‘no phenotype’ genes. First, we showed that phenotype genes are more likely to be disease genes compared to no phenotype genes. Phenotype genes were further divided into ‘embryonic lethal’, ‘postnatal lethal’, and ‘non-lethal phenotype’ groups. Interestingly, embryonic lethal genes, the most essential genes in mouse, were less likely to be disease genes than postnatal lethal genes. These findings indicate that some extremely essential genes are less likely to be disease genes, although human disease genes tend to display characteristics of essential genes. We also showed that, in lethal groups, non-disease genes tend to evolve slower than disease genes indicating a strong purifying selection on non-disease genes in this group. In addition, phenotype and no phenotype groups showed differing types of disease mutations. Disease genes in the no phenotype group displayed a higher frequency of regulatory mutations while those in the phenotype group had more frequent coding mutations, indicating that the types of disease mutations vary depending on gene essentiality. Furthermore, missense disease mutations in no phenotype genes were found to be more radical amino acid substitutions than those in phenotype genes.