Extensive homologous recombination in classical swine fever virus: A re-evaluation of homologous recombination events in the strain AF407339

In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339. We challenged a previous study which suggested only one recombination event in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339, respectively. The first one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF333000","term_id":"13383931","term_text":"AF333000"}}AF333000 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"AY554397","term_id":"49860681","term_text":"AY554397"}}AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF531433","term_id":"22347661","term_text":"AF531433"}}AF531433 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"GQ902941","term_id":"298113017","term_text":"GQ902941"}}GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.     

Upregulation of the novel lncRNA U731166 is associated with migration,invasion and vemurafenib resistance in melanoma

Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis‐related processes. In this sense, we found the intergenic lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 for cancer processes. Additionally, using naïve and vemurafenib‐resistant cell lines and data from a patient before and after resistance, we found that vemurafenib‐resistant samples had a higher expression of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166. Also, we retrieved data from the literature that indicates lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 in metastasis development. We also pointed herein the lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.     

Genetic diversity in relation to heterosis and combining ability in spring wheat

Summary Genetic diversity among ten varieties of spring wheat used as parents in a diallel cross was assessed through multivariate analysis (D²-statistics) and then related to heterosis and SCA effects of their hybrids. The parents fell into three groups. Group I contained the varieties, Nobre, Girua and Carazinho; group II contained Sonalika, Lyallpur and Pitic 62 and group III contained Indus 66, Balaka, Sonora 64rs and MSl. The varieties of group I were good general combiners, while the varieties of group III were poor combiners. Significant heterotic and SCA effects for yield and yield components were observed in the hybrids of the parents belonging to different groups but not in the same group. Genetic divergence between the parents had a positive relationship with heterosis and SCA effects of the hybrids.     

Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates

The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.     

Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate ({"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.     

Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G₂/M or sub-G₁ fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)⁵ (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap₃A) (8), and in vitro studies showed that Ap₃A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k_cat and increases as large as 30-fold in K_m. Mutants with 2–7-fold increases in K_m had significantly reduced apoptotic indices and the mutant with a 30-fold increase in K_m retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with K_m and k_cat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had K_m and k_cat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a K_m ∼60-fold higher and a k_cat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in K_m and k_cat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap₃A complex and enhance tumor suppressor activity (see

Invasive Non-Typhoidal Salmonella Typhimurium ST313 Are Not Host-Restricted and Have an Invasive Phenotype in Experimentally Infected Chickens

Salmonella enterica serovar Typhimurium Sequence Type (ST) 313 is a major cause of invasive non-Typhoidal salmonellosis in sub-Saharan Africa. No animal reservoir has been identified, and it has been suggested that ST313 is adapted to humans and transmission may occur via person-to-person spread. Here, we show that ST313 cause severe invasive infection in chickens as well as humans. Oral infection of chickens with ST313 isolates {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 and Q456 resulted in rapid infection of spleen and liver with all birds infected at these sites by 3 days post-infection. In contrast, the well-defined ST19 S. Typhimurium isolates F98 and 4/74 were slower to cause invasive disease. Both ST19 and ST313 caused hepatosplenomegaly, and this was most pronounced in the ST313-infected animals. At 3 and 7 days post-infection, colonization of the gastrointestinal tract was lower in birds infected with the ST313 isolates compared with ST19. Histological examination and expression of CXCL chemokines in the ileum showed that both {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 (ST313) and 4/74 (ST19) strains caused increased CXCL expression at 3 days post-infection, and this was significantly higher in the ileum of {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 vs 4/74 infected birds. At 7 days post-infection, reduced chemokine expression occurred in the ileum of the {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 but not 4/74-infected birds. Histological analysis showed that {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 infection resulted in rapid inflammation and pathology including villous flattening and fusion at 3 days post-infection, and subsequent resolution by 7 days. In contrast, 4/74 induced less inflammation and pathology at 3 days post-infection. The data presented demonstrate that ST313 is capable of causing invasive disease in a non-human host. The rapid invasive nature of infection in the chicken, coupled with lower gastrointestinal colonization, supports the hypothesis that ST313 is a distinct pathovariant of S. Typhimurium that has evolved to become a systemic pathogen that can cause disease in several hosts.     

Over expression of integrin α 5 β 1 in human hepatocellular carcinoma cell line suppresses cell proliferation in vitro and tumorigenicity in nude mice

Integrin 5 1 and 2 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin 5 1 was decreased and another integrin 6 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin 5 and/or 1 cDNAs were established, and designated 5 1.6-7721, 5.3-7721, and 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin 5 and 1 subunits resulted in the overexpression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in 5.3-7721, 1.6-7721, 5 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with 1.6-7721,and 5 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin 5 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin 5 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Temporal and Spatial Diversity of the Tap Water Microbiota in a Norwegian Hospital

We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.Humans are exposed to and consume large amounts of tap water in their everyday life, with the tap water microbiota representing a potent reservoir for pathogens (8). Despite the potential impact, our knowledge about the ecological diversification processes of the tap water microbiota is limited (4, 11).The aim of the present work was to determine the temporal and spatial distribution patterns of the planktonic tap water microbiota. We compared the summer and winter microbiota from two hospital taps supplied from the same water source. We analyzed 16S rRNA gene clone libraries by using a novel alignment-independent approach for operational taxonomic unit (OTU) designation (6), while established OTU diversity and richness estimators were used for the ecological interpretations.Tap water samples (1 liter) from a high-usage kitchen and a low-usage toilet cold-water tap in Akershus University Hospital, Lørenskog, Norway, were collected in January and July 2006. The total DNA was isolated and the 16S rRNA gene PCR amplified and sequenced. Based on the sequences, we estimated the species richness and diversity, we calculated the distances between the communities, and trees were constructed to reflect the relatedness of the microbiota in the samples analyzed. Details about these analytical approaches are given in the materials and methods section in the supplemental material.Our initial analysis of species composition was done using the RDPII hierarchical classifier. We found that the majority of pathogen-related bacteria in our data set belonged to the class Gammaproteobacteria. The genera encompassed Legionella, Pseudomonas, and Vibrio (Table ​(Table1).1). We found a significant overrepresentation of pathogen-related bacteria in the toilet tap (P = 0.04), while there were no significant differences between summer and winter samples. Legionella showed the highest relative abundance for the pathogen-related bacteria. With respect to the total diversity, we found that Proteobacteria dominated the tap water microbiota (representing 86% of the taxa) (see Table S1 in the supplemental material). There was, however, a large portion (56%) of the taxa that could not be assigned to the genus level using this classifier.

TABLE 1.

Cloned sequences related to human pathogens^a

Evaluation of an exotic maize population adapted to a locality

Summary An exotic Zea mays L. population (Tuxpeno) was adapted to North Carolina conditions by first introducing genes for adaptability from two North Carolina varieties ([(Jarvis X Indian Chief)Tuxpeno]Tuxpeno) including four generations of intermating, and then selecting for adaptability using maturity as the primary measure. The study evaluated selection for adaptability and the diversity available between adapted Tuxpeno and the local varieties, Jarvis and Indian Chief. Analytical procedures were developed to quantify the diversity between populations and the complementation of local varieties by introduced germ plasms. The analyses utilized the specific effects available from the diallel mating design.Three replicate selections responded similarly under simple recurrent mass selection (1/10) for the earliest disease-free plants initially and additionally for plant types (primarily height) in the final generation. The 1/4 local germ plasm permitted rapid adaptation of Tuxpeno gene pool to local conditions. The adapted Tuxpeno populations yielded similarly to the local populations with an average heterosis for grain yield of 28% when crossed to the local populations used as source of genes for adaptability. The diversity found between adapted Tuxpeno lines and these local varieties based on genes affecting grain yield was 1.5 to 2.5 times that measured between the local varieties (Jarvis and Indian Chief). Diversity lost through intergradation with local material was a reasonable investment. Yield genes introduced from Tuxpeno complemented local gene pools through nonadditive, primarily dominance-associated, gene effects. Reassortment of major gene blocks apparently occurred leading to significant divergence among replicate selections involving both additive-associated and dominance-associated gene effects.Paper No. 6355 of the North Carolina Agri. Res. Ser., Raleigh, NC. Research supported in collaboration with the Rockefeller Foundation and CIMMYT, D.F. (Mexico)     

Le syndrome de Klinefelter chez l'enfant

Klinfelter syndrome was first described in adult males with gynecomastia, azoospermia and hypergonadotropic hypogonadism. Children with the 47, XXY karyotype demonstrate few clinical findings so Klinefelter syndrome is rarely diagnosed until adult life. Besides children who have been diagnosed during prenatal genetic testing, in infancy a male with 47, XXY (or variants: 46, XY-47, XXY; 48, XXXY; 48, XXYY, 49, XXXXY) may be found while undergoing evaluation of micropenis, hypospadias, cryptorchidism or facial anomalies. The older child may present with learning disabilities, behavior disorders or tall stature. At the time of puberty, the clinical picture includes small testes, gynecomastia and an eunuchoid habitus. Early diagnosis of Klinefelter syndrome must be performed since it has been demonstrated that early treatment with androgens may ameliorate many aspects of the clinical symptoms and attenuate or prevent behavioral and psychiatric disorders associated with 47, XXY males.     

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded bla_TEM, bla_SHV, bla_CTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7 % of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74 % of K. pneumoniae isolates; with prevalence in the following order: bla_TEM > bla_SHV > bla_CTXM. QNR genes were detected in 67 % samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value = 0.000125). Sequence analysis of some bla_TEM, bla_SHV, bla_CTX-M and QNRB PCR products revealed presence of bla_TEM1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"JN193522","term_id":"343796760","term_text":"JN193522"}}JN193522), bla_TEM116 ({"type":"entrez-nucleotide","attrs":{"text":"JN193523","term_id":"343796762","term_text":"JN193523"}}JN193523 and {"type":"entrez-nucleotide","attrs":{"text":"JN193524","term_id":"343796764","term_text":"JN193524"}}JN193524), bla_SHV11, bla_CTXM72 variants ({"type":"entrez-nucleotide","attrs":{"text":"JF523199","term_id":"329024835","term_text":"JF523199"}}JF523199) and QNRB1 ({"type":"entrez-nucleotide","attrs":{"text":"JN193526","term_id":"343796802","term_text":"JN193526"}}JN193526 and {"type":"entrez-nucleotide","attrs":{"text":"JN193527","term_id":"343796804","term_text":"JN193527"}}JN193527) in our samples.     

Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.Cellulases catalyze the breakdown of cellulose into simple sugars that can be fermented to ethanol. The large amount of natural cellulose available is an exciting potential source of fuels and chemicals. However, the detailed molecular mechanisms of crystalline cellulose degradation by glycoside hydrolases are still not well understood and their low efficiency is a major barrier to cellulosic ethanol production.Thermobifida fusca is a filamentous soil bacterium that grows at 50°C in defined medium and can utilize cellulose as its sole carbon source. It is a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay and produces a set of enzymes that includes six different cellulases, three xylanases, a xyloglucanase, and two CBM33 binding proteins (12). Among them are three endocellulases, Cel9B, Cel6A, and Cel5A (7, 8), two exocellulases, Cel48A and Cel6B (6, 19), and a processive endocellulase, Cel9A (5, 7).T. fusca Cel9A-90 (Uniprot {"type":"entrez-protein","attrs":{"text":"P26221","term_id":"2506384","term_text":"P26221"}}P26221 and {"type":"entrez-protein","attrs":{"text":"YP_290232","term_id":"72162575","term_text":"YP_290232"}}YP_290232) is a multidomain enzyme consisting of a family 9 catalytic domain (CD) rigidly attached by a short linker to a family 3c cellulose binding module (CBM3c), followed by a fibronectin III-like domain and a family 2 CBM (CBM2). Cel9A-68 consists of the family 9 CD and CBM3c. The crystal structure of this species (Fig. ​(Fig.1)1) was determined by X-ray crystallography at 1.9 Å resolution (Protein Data Bank [PDB] code 4tf4) (15). Previous work has shown that E424 is the catalytic acid and D58 is the catalytic base (11, 20). H125 and Y206 were shown to play an important role in activity by forming a hydrogen bonding network with D58, an important supporting residue, D55, and Glc(−1)O1. Several enzymes with amino acid changes in subsites Glc(−1) to Glc(−4) had less than 20% activity on bacterial cellulose (BC) and markedly reduced processivity. It was proposed that these modifications disturb the coordination between product release and the subsequent binding of a cellulose chain into subsites Glc(−1) to Glc(−4) (11). Another variant enzyme with a deletion of a group of amino acids forming a block at the end of the catalytic cleft, Cel9A-68 Δ(T245-L251)R252K (DEL), showed slightly improved filter paper (FP) activity and binding to BC (20).Open in a separate windowFIG. 1.Crystal structure of Cel9A-68 (PDB code 4tf4) showing the locations of the variant residues, catalytic acid E424, catalytic base D58, hydrogen bonding network residues D55, H125, and Y206, and six glucose residues, Glc(−4) to Glc(+2). Part of the linker is visible in dark blue.The CBM3c domain is critical for hydrolysis and processivity. Cel9A-51, an enzyme with the family 9 CD and the linker but without CBM3c, had low activity on carboxymethyl cellulose (CMC), BC, and swollen cellulose (SWC) and showed no processivity (4). The role of CBM3c was investigated by mutagenesis, and one modified enzyme, R557A/E559A, had impaired activity on all of these substrates but normal binding and processivity (11). Variants with changes at five other CBM3c residues were found to slightly lower the activity of the modified enzymes, while Cel9A-68 enzymes containing either F476A, D513A, or I514H were found to have slightly increased binding and processivity (11) (see Table ​Table1).1). In the present work, CBM3c has been investigated more extensively to identify residues involved in substrate binding and processivity, understand the role of CBM3c more clearly, and study the coordination between the CD and CBM3c. An additional goal was to combine amino acid variants showing increased crystalline cellulose activity to see if this further increased activity. Finally, we have investigated whether the changes that improved the activity of Cel9A-68 also enhanced the activity of intact Cel9A-90.

TABLE 1.

Activities of Cel9A-68 CBM3c variant enzymes and CD variant enzymes used to create the double variants

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.