Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5′ or 3′ splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons. Received: 17 July 1997 / Accepted: 28 August 1997     

Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, “c-shaped”, circular and “figure-8” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

An acid and highly thermostable xylanase from <Emphasis Type="Italic">Phialophora</Emphasis> sp. G5

An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1–20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0–9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg⁻¹), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley–soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.     

MTHFD1 G1958A, BHMT G742A, TC2 C776G and TC2 A67G polymorphisms and head and neck squamous cell carcinoma risk

Alterations in folate metabolism may contribute to the process of carcinogenesis by influencing DNA methylation and genomic stability. Polymorphisms in genes encoding enzymes involved in this pathway may alter enzyme activity and consequently interfere in concentrations of homocysteine and S-adenosylmethionine that are important for DNA synthesis and cellular methylation reactions. The objectives were to investigate MTHFD1 G1958A, BHMT G742A, TC2 C776G and TC2 A67G polymorphisms involved in folate metabolism on head and neck cancer risk and the association between these polymorphisms with risk factors. Polymorphisms were investigated in 762 individuals (272 patients and 490 controls) by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and Real Time-PCR. Chi-square and Multiple logistic regression were used for the statistical analysis. Multiple logistic regression showed that tobacco and male gender were predictors for the disease (P < 0.05). Hardy–Weinberg equilibrium showed that the genotypic distributions were in equilibrium for both groups in all polymorphisms studied. The BHMT 742GA or AA genotypes associated with tobacco consumption (P = 0.016) increase the risk for head and neck squamous cell carcinoma (HNSCC). The present study suggests that BHMT 742GA polymorphism associated to tobacco modulate HNSCC risk. However, further investigation of gene–gene interactions in folate metabolism and studies in different populations are needed to investigate polymorphisms and HNSCC risk.     

Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio ♀ × G. rarus ♂) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.     

Analysis of the DNA Marker Specific to the Wheat G Genome

A RAPD marker specific for the G genome of wheat was identified. The corresponding 1171-bp DNA sequence was cloned and analyzed. Screening of the database did not reveal any homologies with the known plant DNA sequences. Using the primers specific to the flanking regions of the marker sequence, PCR analysis of the polyploid wheat species and the diploid species of the section Sitopsis was carried out. In addition, using the cloned sequence as a molecular hybridization probe, RFLP analysis of the genomic DNA of these species was performed.     

Prevalence of carriers of the most common medium-chain acyl-CoA dehydrogenase (MCAD) deficiency mutation (G985A) in The Netherlands

The G985A mutation represents about 90% of all medium-chain acyl-CoA dehydrogenase (MCAD) allele mutations that cause the clinical symptoms of MCAD deficiency. The prevalence of carriers varies between different European populations, with high frequencies in the northwestern part of Europe. To determine the prevalence of MCAD carriers with the G985A mutation in The Netherlands, we collected 6195 Guthrie cards of newborns. Mutation detection was performed with the polymerase chain reaction (PCR), in which a NcoI restriction site was created in the presence of a G985A mutation in the PCR product, followed by NcoI digestion, and gel electrophoresis. We detected a G985A carrier frequency of 1 in 59 (95% CI 1/50–1/73) in The Netherlands. The total prevalence of carriers was estimated to be 1 in 55 (95% CI 1/46– 1/68), based on a relative G985A frequency of 94% in The Netherlands. Received: 18 December 1995 / Revised: 14 February 1996     

Comparative studies on the structure and adhesion of setae in G. gecko and G. swinhonis

Scanning electron microscopy (SEM) and histological techniques were used to observe and study the setae structures of two gecko species (G. gecko and G. swinhonis) and the relationships between these structures and the adhesive forces. The SEM results showed that the setae of these two species were densely distributed in an orderly fashion, and branched with curved tips. The setae of G. gecko had cluster structures, each cluster containing 4-6 setae whose terminal branches curved towards the center of the toes at ~ 10o, the tips of the branches like spatulae and densely arrayed at an interval of less than 0.2―0.3 μm. On the contrary, the branch tips in the setae of G. swinhonis were curled, and the terminal parts of setae curved towards the center of the toes at various angles. Usually the setae of these gecko species branch twice at the top at intervals greater than that of G. gecko. The histological observation found that inside the setae of these two species there were plenty of unevenly distributed contents, such as epithelia, fat cells, pigmental cells and muscle tissue, but no gland cells existed. The results of functional experiments suggested that modifying the structure of gecko's setae could reduce its adhesive ability dramatically, demonstrating the positive correlation between the structure of the gecko's setae and its adhesive ability. The above results provide important information in designing bio-mimic setae and bio-gecko robots.     

A Novel Community of Acidophiles in an Acid Mine Drainage Sediment

A sediment sample (pH 2.5) was collected at an acid mine drainage site in Anhui, China. The present acidophilic microbial community in the sediment was studied with a 16S rRNA gene clone library. Small-subunit rRNA genes were PCR amplified, cloned and screened by amplified rDNA restriction analysis (ARDRA). Subsequently, 10 different clones were identified and they were affiliated with Acidobacteria, β/γ-Proteobacteria, δ-Proteobacteria, Nitrospira, Candidate division TM7, and Low G + C Gram-positives. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the sediment that were mostly novel. Unexpectedly, 16S rRNA gene sequences affiliated with δ-Proteobacteria were found to constitute more than 60% of clone library. To our knowledge, this is the first occasion that bacteria of δ-Proteobacteria have been found dominant in the acidic habitat. Anaerobic sulfate- or metal reduction is the predominant physiological trait of bacteria of this subdivision. The high sulfate, ferric iron and the presence of bioavailable carbon in the anaerobic microenvironment may result in the dominance of bacteria of δ-Proteobacteria.     

Polymerase Chain Reaction in Detection of Gymnodinium mikimotoi and Alexandrium minutum in Field Samples from Southwest India

Polymerase chain reaction (PCR) primers were constructed for the detection of two toxic dinoflagellate species, Gymnodinium mikimotoi and Alexandrium minutum. The primers amplified a product of expected size from cultured cells of G. mikimotoi and A. minutum. The species-specific primers targeting G. mikimotoi did not yield any product with a wide range of other cultured algae used as negative controls. Primers designed for A. minutum were species-group-specific since it PCR yielded a product from the closely related species A. ostenfeldii and A. andersonii, but not from other species of this genus tested. The confirmation of PCR products was performed by digestion of the products with restriction enzymes. Sensitivity analyses of the primers on DNA template from cultured cells was positive by PCR at a DNA template concentration of 1.5 × 10⁻⁴ ng/μl (0.3 cells/L) for A. minutum, and at a DNA concentration of 2.5 × 10⁻² ng/μl (697 cells/L) for G. mikimotoi. The PCR method for detection of G. mikimotoi and A. minutum was applied on field samples collected with a plankton net. Gymnodinium mikimotoi could be detected in 11 field samples by microscopy, and all these field samples were positive by PCR. The cell counts of G. mikimotoi in simultaneously collected water samples ranged from 306 to 2077/L. Alexandrium minutum could be detected by microscopy in 3 different field samples. The cell counts in water samples collected at the same time as the net samples ranged from 115 to 1115 cells/L. Alexandrium minutum was detected by PCR in these field samples, with the exception of the sample displaying the lowest cell count (115 cells/L). Plankton samples that were negative by microscopy for any of the two target species were also negative by PCR. All the PCR products from field samples were confirmed by restriction enzyme digestion. The application of PCR-based detection of harmful algal bloom species for aquaculture and monitoring purposes in natural field samples is discussed. Received April 4, 2000; accepted September 25, 2000.     

The ATPase activity of the <Emphasis Type="Italic">G2alt</Emphasis> gene encoding an aluminium tolerance protein from <Emphasis Type="Italic">Anoxybacillus gonensis</Emphasis> G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with K _m ⁻ and V _max ⁻ values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg²⁺ and Na⁺ ions, while Zn²⁺ and Al³⁺ stimulate the activity. Cd²⁺ and Ag⁺ reduced the activity and Li⁺, Cu²⁺, and Co²⁺ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Correlation Analysis of New Soybean [<i>Glycine max</i> (L.) Merr] Gene Gm15G117700 with Oleic Acid

The fatty acid dehydrogenase gene plays an important role in regulating the oleic acid content in soybean. Genome-wide association study screened out soybean oleic acid related gene Gm15G117700. A fragment size of 693bp was obtained by PCR amplification of the gene and, it was connected by seamless cloning technology to the pMD18T cloning vector. Based on the gene sequence cloned, bioinformatic analysis of gene protein was performed. The overexpression vector of Gm15G117700 and the CRISPR/Cas9 gene editing vector were constructed. The positive plants were obtained by Agrobacterium-mediated transformation of soybean cotyledon nodes and T₂ plants were identified by conventional PCR, QT-PCR and Southern blot hybridization. 10 copies of high and low oleic acid seeds were selected for QT-PCR to identify the expression content of Gm15G117700 gene in different soybeans, and finally near-infrared spectroscopy analyzer was used to identify the oleic acid quality of soybeans. T₂ RT-PCR identification showed that overexpression was reduced by 3.94%, and gene editing was increased by 3.49%. It is determined that the Gm15G117700 gene may belong to a regulatory gene, a minor gene that can promote the conversion to linoleic acid content in soybean oleic acid synthesis. The gene cloning and its functional verification was not reported yet. This is the first report by PCR amplification of soybean Gm15G117700 genes and gene expression vector. Improving the content of oleic acid in soybean lay a foundation for researchers. Therefore;this study clearly identified the function of soybean Gm15G117700 gene and its role played in oleic acid synthesis and metabolism.     

Rapid Real-Time PCR Assay for Culture and Tissue Identification of <Emphasis Type="Italic">Geomyces destructans</Emphasis>: the Etiologic Agent of Bat Geomycosis (White Nose Syndrome)

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS–PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10⁻¹–5 × 10⁷), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)–PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.     

Characterization of clinical isolates of group C and G streptococci on the basis of protein G gene

Treatment of human group C and G streptococci with cyanogen bromide results in solubilization of surface protein G molecules. Strain-to-strain variation in the quanity, molar mass and functional activity of protein G extracted from representative group C and G isolates led to the identification of three structurally and functionally distinct forms of the protein. Using different molecular biological approaches it was possible to determine the group of streptococci (C or G), or the quantity of IgG and HSA domains. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Effects of temperature and salinity on the growth of Gracilaria verrucosa and G. chorda, with the Potential for mariculture in Korea

Effects of temperature and salinity on the growth of the two agarophytes, Gracilaria verrucosa (Hudson) Papenfuss and Gracilaria chorda Holmes were examined in Korea. Both species grew over a wide range of temperatures (10–30 ^∘C) and salinities (5–35‰), and grew well at 17–30 ^∘C and a salinity of 15–30‰. In culture, G. verrucosa grew faster than G. chorda and their maximum growth rates were 4.95% day⁻¹ (30 ^∘C, 25‰) and 4.47% day⁻¹ (at 25 ^∘C, 25‰), respectively. In the field population the maximum growth and fertility of G. chorda were observed in summer. The growth rate of G. verrucosa was slightly higher than that of G. chorda for 2 weeks on the cultivation rope and in culture but it was much lower after being contaminated with epiphytes. The biomass of the epiphytes was 0.82 g dry wt. per host plant in G. verrucosa and 0.001 g in G. chorda. G. chorda exhibited resistance to epiphytism and grew 7 times in length and the dry weight increased 15 times after 55 days. In conclusion, G. chorda appears to be a good agarophyte with a fast growth rate and resistance to epiphytesm, and compared with G. verrucosa, has good potential for commercial cultivation.     

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra—chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998