Gene Cloning,Expression, and Characterization of the <Emphasis Type="Italic">Geobacillus Thermoleovorans</Emphasis> CCR11 Thermoalkaliphilic Lipase

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.     

Barley β-Galactosidase: Structure, Function, Heterogeneity, and Gene Origin

Barley (Hordeum vulgare) β-galactosidase is composed of a large (45 kDa) and a small (33 kDa) polypeptide. N-terminal sequencing of the polypeptides and antibody reactivity data place the barley enzyme and heterodimeric plant β-galactosidases from jack bean, maize, and wheat in family 35 of the glycosyl hydrolases. Sequence analysis indicates the existence of a subfamily of genes coding for polypeptide precursors that are cleaved to produce the two subunits in heterodimeric β-galactosidases. The heterogeneity of the barley holoenzyme is related, but not restricted, to the N-glycosylation of the small polypeptide. Both polypeptides are essential for the catalytic activity of the enzyme.     

An Immune-Related Gene Evolved into the Master Sex-Determining Gene in Rainbow Trout, Oncorhynchus mykiss

Since the discovery of Sry in mammals [1, 2], few other master sex-determining genes have been identified in vertebrates [3-7]. To date, all of these genes have been characterized as well-known factors in the sex differentiation pathway, suggesting that the same subset of genes have been repeatedly and independently selected throughout evolution as master sex determinants [8, 9]. Here, we characterized in rainbow trout an unknown gene expressed only in the testis, with a predominant expression during testicular differentiation. This gene is a male-specific genomic sequence that is colocalized along with the sex-determining locus. This gene, named sdY for sexually dimorphic on the Y?chromosome, encodes a protein that displays similarity to the C-terminal domain of interferon regulatory factor 9. The targeted inactivation of sdY in males using zinc-finger nuclease induces ovarian differentiation, and the overexpression of sdY in females using additive transgenesis induces testicular differentiation. Together, these results demonstrate that sdY is a novel vertebrate master sex-determining gene not related to any known sex-differentiating gene. These findings highlight an unexpected evolutionary plasticity in vertebrate sex determination through the demonstration that master sex determinants can arise from the de novo evolution of genes that have not been previously implicated in sex differentiation.     

Presence of a Bacterial-Like Citrate Synthase Gene in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>: Recent Lateral Gene Transfers (LGT) or Multiple Gene Losses Subsequent to a Single Ancient LGT?

Citrate synthase is the initial enzyme in the tricarboxylic acid cycle of mitochondria. In plants and fungi, it is the second isozyme in the glyoxylate cycle of peroxisomes (or glyoxysomes), and it is also present in bacteria. Some of the biochemical reactions in the glyoxylate cycle of the ciliated protozoan Tetrahymena pyriformis depend upon mitochondrial enzymes, as T. pyriformis lacks some glyoxysome-specific enzymes. Here we demonstrate a new citrate synthase gene from Tetrahymena thermophila that is different from the mitochondrial counterpart. A potential peroxysome-targeted signal was detected in the N-terminus, suggesting the localization of the enzyme in peroxysomes. Phylogenetic analysis placed the Tetrahymena sequence in a clade consisting of a few sequences from eukaryotes such as cellular slime molds and two land plants, near a green sulfur bacterium and many proteobacteria as a sister group but not in a mitochondrial clade. Southern blot analysis revealed that this type of gene was absent from distantly related ciliates and other species of Tetrahymena except for the closest species, T. mallaccensis. The scattered presence of the bacterial-like genes among distantly related eukaryotes suggests three alternative interpretations of acquisition of the novel glyoxysomal citrate synthase gene via lateral gene transfer (LGT). (1) Some eukaryotes independently acquired the gene from a common bacterium or closely related bacteria via LGT. (2) A hypothetical eukaryote once acquired the gene, which was thereafter independently transferred from the eukaryote to other eukaryotes. (3) A single event of LGT (or duplication) occurred in a certain common ancestor of eukaryotes, followed by multiple losses in many eukaryotic lineages during the subsequent evolution. Considering the monophyly of the bacterial-like eukaryotic citrate synthase genes, the first model is somewhat unlikely, even though it is not impossible. The second and third models can rationally explain the present observation, so these models are discused in some detail.     

Gene cloning,expression, and biochemical characterization of an alkali-tolerant β-mannanase from <Emphasis Type="Italic">Humicola</Emphasis><Emphasis Type="Italic">insolens</Emphasis> Y1

In this article, we firstly report a highly alkali-tolerant fungal β-mannanase from Humicola insolens Y1. The full-length cDNA of the β-mannanase, designated as man5A, has an open reading frame of 1,233 bp that encodes a 411-amino acid polypeptide (Man5A) with a calculated molecular mass of 42.3 kDa. The deduced sequence of Man5A comprises a putative 20-residue signal peptide and a catalytic domain belonging to glycoside hydrolase family 5, and displays 61–85% identities with hypothetical proteins and 32–39% with experimentally verified fungal β-mannanases. Purified recombinant Man5A produced by Pichia pastoris has a specific activity of 1,122 U mg⁻¹ and exhibits optimal activity at pH 5.5 and 70°C. Distinct from other reported fungal β-mannanases, Man5A is highly alkali tolerant, exhibiting 45 and 36% of the maximal activity at pH 8.0 and 9.0, respectively, and more than 10% activity even at pH 10.0. Moreover, Man5A has excellent pH stability at pH 5.0–12.0 and is highly thermostable at 50°C. The higher frequency of alkaline amino acids (Arg and Lys), greater pKa values of the catalytic residues, and more positively charged residues on the surface of Man5A might be the causes. Man5A has strong resistance to various neutral and alkaline proteases, retaining more than 97% of the activity after proteolytic treatment for 1 h. The superior characteristics of Man5A make it more advantageous for the application in the kraft pulp industry.     

Functional Expression of <Emphasis Type="Italic">Spirulina</Emphasis>-Δ<Superscript>6</Superscript> Desaturase Gene in Yeast, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ⁶ desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ⁶-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Δ⁶-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Δ⁶ desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ⁶ desaturation in yeast is more likely to be cytochrome b₅, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.     

Inflammatory,Apoptotic, and Survival Gene Signaling in Alzheimer’s Disease

Aging is associated with an enhanced susceptibility to brain dysfunction, loss of memory, and cognitive decline and significantly influences the quality of life for the affected individual. Recent molecular–genetic approaches have provided powerful insights into common age-related diseases that are both progressive and multifactorial, such as Alzheimer’s disease (AD), and in vitro in AD models. These investigations have uncovered consistent deficits in brain gene signaling mechanisms and neurotrophic substances known to contribute to normal brain function. Inflammatory signaling pathways involving up-regulation of cytosolic phospholipase A₂ and the arachidonic acid cycle, the depletion of the brain-essential fatty acid docosahexaenoic acid (DHA) and DHA-derived neuroprotectin D1, and changes in the expression of key proapoptotic and antiapoptotic members of the Bcl-2 gene family are thought to be major contributors to pathogenic processes in degenerating brain tissue. This review will focus on the roles of stress genes, apoptosis-related genes, and inflammation in the molecular genetics of AD with emphasis on the interactive nature of inflammatory, neurotrophic, and apoptotic signaling and will highlight areas of rapid progress in the characterization of action of DHA and neuroprotectin D1 and address important research challenges. We also attempt to integrate these molecular, genetic, and neurochemical changes with cellular pathways involved in brain aging to formulate an integrated understanding of multifactorial age-related neurologic disease and pharmacotherapeutic strategies that may be useful in the restoration of homeostatic brain function.     

The β-esterase Gene Cluster of Drosophila melanogaster: is ψEst-6 a Pseudogene, a Functional Gene, or both?

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.