Digested PCR Products Research Articles

16A system of molecular markers to identify alleles of the Rht-B1 and Rht-D1 genes controlling reduced height in bread wheat.

Mutant alleles of the Rht-B1 and Rht-D1 (Reduced height) genes are widely used in bread wheat breeding for the development of intensive-type cultivars. These genes and their f lanking regions have been sequenced and the point mutations leading to the nonsense codons (Rht-B1b, Rht-B1e, Rht-B1p and Rht-D1b alleles) and various insertions (Rht-B1c, Rht-B1h and Rht-B1i-1) associated with a change in plant height have been described. DNA-markers based on the allele-specif ic PCR have been developed to identify single-nucleotide changes. However, the use of such technique imposes stringent PCR conditions, and the resulting data are not always unambiguous. An alternative can be found in the CAPS technology: it detects differences in sequences by digesting PCR products. In the absence of restrictases capable of digesting DNA at the point mutation site, restriction sites can be introduced into the primer sequence (derived CAPS). The aim of this study was to propose a system of CAPS-, dCAPS- and STS-markers for identifying alleles of the reduced height genes frequently used in breeding programs. Three CAPS have been developed to identify the Rht-B1b, Rht-D1b, Rht-B1p alleles, as well as two dCAPS for Rht-B1b, Rht-B1e. STS-markers for the insertion-containing alleles Rht-B1c, Rht-B1h and Rht-B1i-1 have been selected from publications. The proposed markers were tested during the genotyping of 11 bread wheat accessions from the VIR collection with the abovementioned mutant alleles and the wild-type Rht-B1a and Rht-D1a. The presence of nonsense mutations was also conf irmed by the results of allele-specif ic PCR. This marker system, along with the existing ones, can be used to identify dwarf ing alleles of the Rht-B1 and Rht-D1 genes in bread wheat for genetic screening of accessions from ex situ collections and/or for marker-assisted selection.

The Association Study between Tumor Necrosis Factor- Receptor 1 36 A/G (TNFR1 36 A/G) Gene Polymorphism and Azoospermic Infertile Men in Erbil City

   Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine that controls cellular activities related to spermatogenesis. (TNFR1) affects TNF- α activity and also may leads to gene dysfunction and male infertility. This retrospective study was done to assess the   linkage of TNFR1 36 A/G gene polymorphism with idiopathic azoospermia in Erbil City. The present study was carried out on 98 infertile, 109 fertile males as healthy control. TNFR1 36 A/G gene polymorphisms were detected in all study subjects, and their seminal fluids were analyzed.   Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is used for identify polymorphism in both cases and control groups. MspA1I restriction enzyme were  digested PCR products. Digested and PCR products were analyzed by 3% gel electrophoresis for 45 minutes at 97 V.   . The results of the present study showed that the incidence of AG genotype in azoospermia male higher than control groups (OR=2.069(1.154-3.708), P=0.014, this study found that G allele frequency in azoospermia men had a significant difference compared to the control group (OR=1.487 (0.992-2.170).  Moreover, AG genotype and G allele associated with an increased risk of non-obstructive azoospermia in Erbil City.

Prevalence of CYP2C19 and ITGB3 polymorphisms among Bangladeshi patients who underwent percutaneous coronary intervention.

Introduction:Antithrombotic agents are the basic therapeutic option for patients with arterial thrombosis who underwent percutaneous coronary intervention (PCI). In Bangladesh, aspirin and clopidogrel are frequently prescribed as antithrombotics or platelet inhibitors. Studies reported the genetic polymorphisms of CYP2C19*2, CYP2C19*17, and ITGB3 cause an alteration of the pharmacodynamic and pharmacokinetic profile of aspirin and clopidogrel. Therefore, we aimed to assess the prevalence of CYP2C19*2, CYP2C19*17, and ITGB3 polymorphisms among Bangladeshi patients with cardiovascular disease (CVD) who underwent PCI.Methods:Here we assessed a total of 1,000 CVD patients (male 782 and female 218) who underwent PCI and were treated with clopidogrel and/or aspirin. We performed genotyping of patients treated with clopidogrel and aspirin by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) methods. The PCR products of clopidogrel-treated patients were screened with agarose gel electrophoresis and then digested with SmaI and NsiI-HF for CYP2C19*2 and CYP2C19*17, respectively. We genotyped aspirin-treated patients with T-ARMS-PCR for missense rs5918 (PlA1/A1) polymorphism of the ITGB3 gene. Then we ran the digested PCR products on 2% agarose gel electrophoresis to detect the mentioned polymorphisms.Results:Among the clopidogrel-treated patients, we observed 64.1% polymorphism (hetero + mutant) of CYP2C19*2 (loss-of-function allele) and 22.7% (hetero + mutant) of CYP2C19*17 (gain-of-function allele). On the other hand, among the aspirin-treated patients, polymorphisms of ITGB3 were 84.1% homozygous (PlA1/A1), 15.6% heterozygous (PlA1/A2), and 0.3% mutant homozygous.Conclusion:In the present study, we observed a high prevalence of genetic polymorphisms of CYP2C19 and ITGB3 genes. Therefore, we recommend genotyping of CVD patients before prescribing clopidogrel or aspirin to prevent coagulation. Based on the genotyping study, the adjustment of doses or alternative generics might require to avoid therapeutic failure or toxicity in some cases.

Investigation of GH and GHRAlu I gene polymorphisms on meat yields in Anatolian water buffalo breed using PCR-RFLP method

The Anatolian water buffalo is a native water buffalo breed only being reared in Turkey. The objective of this study was to investigate gene polymorphisms on exons 4 and 5 of the growth hormone (GH) and on exon 10 of the growth hormone receptor (GHR), which are thought to be related to meat yields of Anatolian water buffalo. Blood samples of 192 water buffaloes from Sivas Province were used in DNA extractions by phenol-chloroform method. DNA samples were amplified by using specific primers in PCR. PCR products were digested by Alu I (GH, n: 167) and Alu I (GHR, n: 192) restriction enzymes in order to determine polymorphisms. Digested PCR products were separated in 2.5%-3% agarose gel electrophoresis to determine allelic polymorphisms. As a result, the LL (78.44%) and LV (21.56%) genotypes and L (0.89) and V (0.11) alleles for the GH gene and the AA (7.81%) and AG (92.19%) genotypes and A (0.54) and G (0.46) alleles for the GHR gene were obtained. Gene polymorphisms were not detected (P > 0.05) on the GH gene, but a significant difference was found for the GHR gene (P < 0.001). Therefore, it can be said that Anatolian water buffaloes have increased meat yield due to the presence of genotypes in gene regions. This was the first investigation of enzyme polymorphisms of the GHR gene in Anatolian water buffaloes.

SCN5A (G892A) gene is associated with AV block conduction disorder in north Indian population

Objective- Electrical abnormalities of the heart cause the cardiac arrhythmia with or without essential structural heart disease. Due to genetic variation, cardiac arrhythmia can occur in any age group patients as well as in healthy people. The present study aimed to examine the allele and genotype of SCN5A (G892A) gene in cardiac conduction disorder (especially AV block conduction disorder) cases and comparison with healthy controls. Method- A total number of 104 cases and 104 controls were enrolled in this study. DNA was extracted using salting out method followed by polymerase chain reaction amplification and restriction endonuclease digestion (using AluI restriction enzyme). Digested PCR products were identified using agarose gel electrophoresis and stained with ethidium bromide. Results- There was a strong association of SCN5A G892A polymorphisms (GA vs. GG, OR= 4.01 and AA vs. GG OR= 3.12) with cardiac conduction disorder. Further multivariate logistic regression analysis, after adjustment for age, gender showed that when compared with wild type GG genotype, carriers of the A alleles had an increase risk of cardiac conduction disorder (OR-1.89, 95%CI- 0.78-1.67, p value- <0.0001). Conclusion- In conclusion the results of the present study suggest that the SCN5A G892A gene polymorphism carries an increased risk for cardiac conduction disorder in north Indian populations.

Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics.

Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of P. furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

Influence of APOA5 (rs662799 and rs3135506) gene polymorphism in acute myocardial infarction patients and its association with basic coronary artery disease risk factors

The aim of this study was to examine the allele and genotype of APOA5 -1131T/C (rs662799) and APOA5-56C/G (rs3135506) gene in acute myocardial (AMI) case and control subjects. 304 case and 304 controls were enrolled in this study. DNA was extracted using salting out method followed by polymerase chain reaction amplification and restriction endonuclease digestion (using MseI for -1131T/C and Taq1 for -56C/G). Digested PCR products were identified using agarose gel electrophoresis and stained with ethidium bromide. There was a strong association between APOA5 -1131T/C (TC vs. TT, OR= 1.58 and CC vs. TT OR= 2.43) and APOA5 -56C/G (CG vs. CC, OR= 1.64 and GG vs. CC, OR= 2.44) polymorphisms with AMI. Out of the six potential risk factors for coronary artery disease, only smoking, diabetes and hypertension were found to be associated with APOA5 gene and increased the risk of AMI. Smoking was the most prominent risk factor for both the genes. Other risk factors like history of dyslipidemia, obesity and family history of coronary artery disease did not reveal any potential association with the candidate gene. Our data demonstrate that both the SNPs in the APOA5 gene (-1131T/C, and -56C/G) were strongly associated with AMI in north Indian population.

Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond.

The genus Bacillus comprises of a diverse group with a wide range of nutritional requirements and physiological and metabolic diversity. Their role in nutrient cycle is well documented. 16S rDNA sequences do not always allow the species to be discriminated. In this study 40 Bacillus spp. obtained from fish culture pond and 10 culture type strains were analysed for their genomic diversity by PCR–RFLP of intergenic spacer region of 16S-23S and HSP60 genes. TaqI digestion of PCR products amplified by ITS PCR did not render distinctive RFLP patterns. Numerical analysis of ITS PCR–RFLP pattern differentiated the isolates into 11 clusters. Same species were found to be grouped in different clusters. But PstI digested PCR products amplified from HSP60 gene of the isolates showed distinctive RFLP patterns. The dendrogram constructed from HSP60 PCR–RFLP delineated the isolates into 11 clusters also. All the clusters, except cluster I grouped only one type of species. The results showed that Bacillus spp. could be clearly distinguished by PCR–RFLP of HSP60 gene. Therefore, the HSP60 gene is proposed as an additional molecular marker for discrimination of Bacillus group.

Development of Functional Markers for Detection of Inactive DFR-A Alleles Responsible for Failure of Anthocyanin Production in Onions (Allium cepa L.)

Inactivation of the gene coding for dihydroflavonol 4-reductase (DFR) is responsible for the color difference between red and yellow onions (Allium cepa L.). Two inactive DFR-A alleles, DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX>, were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-<TEX>$A^{PS}$</TEX> allele. A derived cleaved amplified polymorphic sequences (dCAPS) primer was designed to detect the single nucleotide polymorphism, an A/T transition, which produced the premature stop codon. Digested PCR products clearly distinguished the homozygous and heterozygous red <TEX>$F_2$</TEX> individuals. Meanwhile, to develop a molecular marker for detection of the DFR-<TEX>$A^{DEL}$</TEX> allele in which entire DFR-A gene was deleted, genome walking was performed and approximately 3 kb 5' and 3' flanking sequences of the DFR-<TEX>$A^R$</TEX> coding region were obtained. PCR amplification using multiple primers binding to the extended flanking regions showed that more of the extended region of the DFR-A gene was deleted in the DFR-<TEX>$A^{DEL}$</TEX> allele. A dominant simple PCR marker was developed to identify the DFR-<TEX>$A^{DEL}$</TEX> allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX> alleles in yellow onion cultivars bred in Korea and Japan was surveyed using molecular makers developed in this study. Results showed predominant existence of the DFR-<TEX>$A^{PS}$</TEX> allele in yellow onion cultivars.

Role of tumor necrosis factor gene single nucleotide polymorphisms in the natural course of 2009 influenza A H1N1 virus infection

To identify the role of single nucleotide polymorphisms (SNPs) of the tumor necrosis factor (TNF) gene in the natural course of 2009 influenza A H1N1 virus infection. Genomic DNA was isolated from 109 patients with an H1N1 infection and from 108 healthy volunteers. SNPs of the TNF gene were assessed after electrophoresis of the digested PCR products by restriction enzymes. The frequency of the -238 A allele was significantly greater among patients than among controls. Viral pneumonia developed in 20 of 96 non-carriers of at least one -238 A allele (20.8%) and in seven of 13 carriers of at least one -238 A allele (53.8%, p=0.016). Logistic regression analysis showed that the most important factors associated with the development of pneumonia were the presence of an underlying disease (p=0.021, odds ratio (OR) 3.08) and the carriage of at least one -238 A allele (p=0.041, OR 3.74). Gene transcripts of the TNF gene were greater among non-carriers of the -238 A allele than among carriers of the -238 A allele. The -238 A SNP allele of the TNF gene imposes on the course of 2009 H1N1 virus infection and is an independent risk factor for pneumonia.

A cleaved amplified polymorphic sequence (CAPS) method for the identification of geographical isolates of Schistosoma japonicum in China.

Schistosomiasis japonica, caused by the blood fluke Schistosoma japonicum, is a severe parasitic zoonosis, causing significant morbidity, mortality and considerable economic losses in south-east Asia, particularly in mainland China (Zhou et al., 2005; Bergquist et al., 2008; Zhou et al., 2008; Li et al., 2010). Although there have been significant efforts to control schistosomiasis in China, this disease appears to be re-emerging due to climate changes and human activities (Wang et al., 2009). Thus far, schistosomiasis is still endemic in seven provinces (Yunnan, Sichuan, Anhui, Hubei, Jiangxi, Jiangsu and Hunan provinces) of China (Ross et al., 2001), and the impact of this communicable disease in China is almost equal to that of HIV/AIDS and tuberculosis (Engels et al., 2005). In previous studies, genetic variation among S. japonicum populations from different endemic provinces in China has been detected using a variety of genetic markers (Bogh et al., 1999; Sorensen et al., 1999; Zhu et al., 1999; Shrivastava et al., 2005; Zhao et al., 2009a; Zhao et al., 2009b; Zhao et al., 2009c). However, these markers and the detected genetic variability have not been utilized for the practical identification and differentiation of S. japonicum geographical isolates in China. The economic reform and the deterioration of ecological environment, flash floods, landslides and the mobility of human population appear to be having an impact on the transmission of S. japonicum (Li et al., 2000; Seto et al., 2008). Consequently, it is important to be able to identify different genetic variants of S. japonicum in China. Various methods for the analysis of genetic variation have been developed, including single-strand conformation polymorphism (Simoes et al., 2007), denaturing high-performance liquid chromatography (Nickerson et al., 2000), gene chips (Schmalzing et al., 2000), TaqMan probe (Shi et al., 1999) and pyrosequencing technology (Ahmadian et al., 2000). However, such techniques can be time-consuming and/or require detection instruments or labelled oligonucleotides. In contrast, the cleaved amplified polymorphism sequence (CAPS) technique is a simple and effective method to allow the rapid detection of single nucleotide polymorphisms (SNPs) (Neff et al., 1998; Komori and Nitta, 2005). CAPS is based on the PCR-based amplification of targeted genes and subsequent digestion of amplicons using restriction enzymes. The digested PCR products are separated by agarose gel electrophoresis. CAPS generates the same type of data as traditional restriction fragment length polymorphism analysis, but significantly reduces the amount of purified DNA required for analysis and time-consuming steps. This method can detect both homozygous and heterozygous individuals (Weiland and Yu, 2003; Komori and Nitta, 2005). CAPS has been successfully used to study genetic diversity in plants, such as Arabidopsis thaliana (Hardtke et al., 1996; Barth et al., 2002), Cryptomeria japonica (Tsumura and Tomaru, 1999; Tsumura et al., 1999) and Pisum sativum L. (Konovalov et al., 2009), but has seldom been used to study variation in parasites (Gandhi et al., 2009). The aims of the present study were to identify SNP sites specific for geographical isolates of S. japonicum based on results of previous studies. Using these SNPs, the CAPS technique was developed and used to identify and differentiate S. japonicum isolates from Yunnan province and those from other endemic provinces.

Genetic polymorphism BMP15 and GDF9 genes in Sangsari sheep of Iran

Different mutations in the bone morphogenetic protein 15 (BMP15) and the Growth Differentiation Factor 9 (GDF9) genes cause increased ovulation rate and infertility in a dosage-sensitive manner in sheep. In this study, blood samples (140 ewes and 10 rams) were initially taken from 150 Sangsari sheep breed in Damghan animal breeding Centre using venojects treated with the anti-clot substance (EDTA) and subsequently their DNA content were salted out and extracted. Using two pairs of specific primers, two DNA fragments were amplified from exon 1 of GDF-9 (462 bp) and exon 2 of BMP15 (141 bp) genes. The resulted PCR products were digested using HhaІ and HinfI restriction enzymes for GDF9 and BMP15 genes, respectively. Digested PCR products with HhaІ enzyme showed a G to A substiuation in GDF9 locus. The wild type allele of this gene (G/+) with two restriction site resulted DNA fragments of 156.52 and 254 bp while the mutant allele (G/-) with one restriction site resulted two DNA fragments with the size of 52 and 410 bp. Genotype frequencies for G (+/+),G (+/-) and G (-/-) were 70.72, 36.88 and 1.40%, respectively. Restriction digested of PCR products for BMP15 locus with Hinf I enzyme showed C to T transition. BMP15 luci was not polymorphic. From studied luci, only GDF9 was polymorphic in Iranian Sanghsari sheep. Key words: PCR, polymorphism, GDF9, BMP15, Sangsari sheep.

Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion

Hormonal, inflammatory and vascular alterations during pregnancy are thought to be involved in pregnancy loss. The role of progesterone, the actions of which involve cytokines and vascular endothelial growth factor (VEGF), has been thoroughly studied. Genetic factors are involved in modulation of these molecules, and several genes have been associated with recurrent spontaneous abortion (RSA), as well other obstetric conditions. Here we investigate the relationship between RSA and gene polymorphisms of the progesterone receptor (PROGINS), interleukin-1 receptor 1 (PstI) and vascular endothelial growth factor (VEGF) (-634, 936). This case-controlled study comprised a total of 89 women with idiopathic RSA and a control group of 191 women with at least two successful pregnancies and no miscarriages. Genomic DNA was extracted from whole blood, and polymorphism genotyping was conducted by digesting PCR products with specific restriction endonucleases. No correlations were found in any of the investigated polymorphisms, even among dominant, co-dominant and additive inheritance models and alleles. Our results support the hypothesis that these specific gene polymorphisms are not the major determinant of pregnancy success. Although we did not find any correlations, the investigation of these and other polymorphisms remains a challenge in the evaluation of RSA.

Lack of Association BetweenFXIII-Val34Leu,FVII-323 del/ins, and Transforming Growth Factor β1 (915G/T) Gene Polymorphisms and Bronchopulmonary Dysplasia: A Single-Center Study

Bronchopulmonary dysplasia (BPD) is a multifactorial disease of preterm infants that is characterized by airway injury, inflammation, and parenchymal remodeling. Extravascular fibrin deposits in septae and alveoli due to the altered fibrin turnover are the pathological hallmarks of BPD that strongly indicates the importance of the imbalance in the competing activities of coagulation and fibrinolysis. Activation of the coagulation cascade leads to intraalveolar fibrin deposition in many inflammatory pulmonary disorders. Increased fibrin formation or decreased fibrinolysis may cause extravascular fibrin deposition. We evaluated the association between FXIII-Val34Leu, FVII-323 del/ins, and transforming growth factor beta1 (TGF-beta(1)) (915G/T) gene polymorphisms in patients with BPD. The study group consisted of 98 preterm infants with BPD. Ninety-four of the 192 preterm neonates were without BPD and sampled for the control group. Restriction fragment size analyses were performed by examining digested PCR products for FXIII-Val34Leu, FVII-323 del/ins, and TGF-beta(1) (915G/C) genotypes. No significant associations were found between FXIII-Val34Leu, FVII-323 del/ins, TGF-beta(1) (915G/C) gene polymorphisms and BPD phenotype in our population. Further studies with other genes are required for the identification of molecular predisposing factors for BPD that may help in the development of new treatments and hence might allow for targeting of this treatment to a "high-risk" subgroup, reducing unnecessary exposure to potentially harmful therapies.

Relationship between PON1L55M and Q192R gene polymorphisms and high APO B/APO A-I ratios

Elevated Apolipoprotein B Apolipoprotein A-I ratio is a risk factor for predicting coronary artery disease (CAD). Paraoxonase 1 (PON1) is a high density lipoprotein (HDL) associated serum enzyme. PON1 protects lowdensity lipoproteins (LDLs) from oxidative modifications and thus has a protective effect against CAD progression. There are two common polymorphisms, Q192R and L55M, in PON1 gene. There may be a relationship between these polymorphisms and elevated ApoB/ApoA-I ratios. Therefore, we decided to evaluate effect of these polymorphisms on individuals with high and normal ApoB/ApoA-I ratios. To evaluate Q192R and L55M polymorphisms in Iranian case group (n=75) with high ApoB/ApoA-I ratio, and control group (n=75) with normal ApoB/ApoA-I ratio, we carried out PCR using specific primers. Then, we digested PCR products by RFLP. ApoB and ApoA-I levels were determined by immunoturbidimetry method. Genotype frequencies for Q192R were determined: 49.3%QQ, 44%QR, 6.7%RR in case group, and 53.3%QQ, 33.3%QR, 13.4%RR in controls (P= 0.236). Genotype frequencies for L55M were determined: 21.3%LL, 68%LM, 10.7%MM in case group and 42.7%LL, 52%LM, 5.3%MM in controls (P= 0.016). A significant relationship between L55M polymorphism and familial history of cardiovascular disease was found (P= 0.011). In our study PON1L55M polymorphism was associated with high ApoB/ApoA-I ratios in case group. Thus, L55M polymorphism may be an independent risk factor for cardiovascular disease. Since L55M polymorphism was associated with familial history of cardiovascular disease, it is better to evaluate L55M polymorphism in younger ages even in the absence of high ApoB/ApoA-I ratios.

Construction of a New Fusion Protein Vector Associated to Fibronectin Binding Protein A and Clumping Factor A Derived from Staphylococcus aureus NCTC8325

Objective(s) Staphylococcus aureus is a leading cause of many nosocomial and community acquired infections. According to many reports, antibiotic therapy can not guarantee the eradication of S. aureus infections. Thus designing an adhesin based vaccine could restrain the S. aureus infections. This study designed for construction of a new fusion protein vaccine against S. aureus infections based on adhesin molecules fibronectin binding protein A (FnBPA) and clumping factor A (ClfA). Materials and Methods Bioinformatic experiments were performed using Oligo analyzer and DNAMAN softwares. The fragments corresponding to fnbA binding domain and a C-terminal fragment from clfA were amplified from S. aureus NCTC8325 genomic DNA. Purified PCR products and the vector, pET15b, were digested with NcoI and BamHI. The digested PCR products were hybridized together and then ligated to digested vector. Finally incomplete construct was assembled by Taq DNA polymerase. To quick confirmation of cloning procedure the new construct designated pfnbA-clfA was digested with NcoI and BamHI. To further verification, the product was sent for sequencing. Results The data based on bioinformatic analysis showed no homology between fusion protein and human proteins. Digestion of new vector with NcoI and BamHI confirmed the ligation of fusion protein sequence into pET15b. Sequencing results verified the integrity of target sequences. Conclusion This study is the first effort to construct a new fusion protein vector based on S. aureus adhesins using a new design. This project is being continued to study the expression and biological activity of the fusion protein in a cell culture model.

Molecular characterization of Pina and Pinb allelic variations in Xinjiang landraces and commercial wheat cultivars

Grain hardness plays an important role in determining both milling performance and quality of the end-use products produced from common or bread wheat. The objective of this study was to characterize allelic variations at the Pina and Pinb loci in Xinjiang wheat germplasm for further understanding the mechanisms involved in endosperm texture formation, and the status of grain texture in Chinese bread wheat. A total of 291 wheat cultivars, including 56 landraces, and 95 introduced and 140 locally improved cultivars, grown in Xinjiang, were used for SKCS measurement and molecular characterization. Among the harvested grain samples, 185 (63.6%), 40 (13.7%), and 66 (22.7%) were classified as hard, mixed and soft, respectively. Eight different genotypes for the Pina and Pinb loci were identified, including seven previously reported genotypes, viz., Pina-D1a/Pinb-D1a, Pina-D1a/Pinb-D1b, Pina-D1b/Pinb-D1a, Pina-D1a/Pinb-D1p, Pina-D1a/Pinb-D1q, Pina-D1a/Pinb-D1aa, Pina-D1a/Pinb-D1ab, and a novel Pinb allele, Pinb-D1ac. This new allele, detected in Kashibaipi (local landrace) and Red Star (from Russia) has a double mutation at the 257th (G to A substitution) and 382nd (C to T substitution) nucleotide positions of the coding region. Pina-D1b, Pinb-D1b, and Pinb-D1p were the most common alleles in Xinjiang wheat germplasm, with frequencies of 14.3%, 38.1% and 28.6% in hard textured landraces, 25.5%, 56.9% and 11.8% in hard introduced cultivars, and 24.8%, 47.8% and 26.5% in hard locally improved cultivars, respectively. The restriction enzymes ApaI, SapI, BstXI and SfaNI were used to identify Pinb-D1ab or Pinb-D1ac, Pinb-D1b, Pinb-D1e and Pinb-Dg, respectively, by digesting PCR products of the Pinb gene. The unique grain hardness distribution in Xinjiang bread wheat, as well as the CAPs markers for identification of the Pinb alleles provided useful information for breeding wheat cultivars with optimum grain textures.

The Non-catalytic N-terminal Extension of Formylglycine-generating Enzyme Is Required for Its Biological Activity and Retention in the Endoplasmic Reticulum

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.

Relationship between small-for-gestational age births and maternal thrombophilic mutations

Small gestational age (SGA) is one of the major causes of fetal mortality and morbidity. Altered maternal homeostasis as a result of point mutations in the coagulation cascade has been reported as an important risk factor for this adverse pregnancy outcome. This study aims to investigate the relationship between mother's thrombophilic mutations and SGA deliveries in our population. The study group was consisted of sixty-six women who gave birth to one or more SGA babies. 104 women who gave birth to appropriate-for-gestational age (AGA) babies were sampled for the control group. Restriction fragment size analysis were performed by visualizing digested PCR products for Factor V Leiden (G1691A), Factor V Cambridge (A1090G), Factor V A1299G, prothrombin G20210A, methylene tetrahydropholate reductase C677T, A1298C and T1317C mutations. The results of this study indicate that maternal C677T (p=0.01) and A1298C (p<0.01) mutations in MTHFR gene may be suggested as risk factors for SGA outcome in our population. Therefore, maternal screening of these two mutations in the first trimester of pregnancy could help in the assessment of patients.

Analysis of DNA corresponding to group A streptococcal A- and B-regions of M protein by restriction fragment length polymorphism

The emm gene amplicons encoding the N- and partial C-terminal regions of the M protein from Group A streptococcus (GAS) isolates were amplified by PCR and digested with a restriction endonuclease, AluI. Digested PCR products were then resolved by polyacrylamide gel electrophoresis. Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products. Digested with AluI, they exhibited different restriction RFLP patterns. Of 106 GAS isolates examined from normal population and patients with GAS-associated complications, 95 isolates showed RFLP patterns that corresponded to the 13 known M types. The other 11 isolates demonstrated different RFLP patterns other than the known M types. They were then analyzed by DNA sequencing and other six M types were obtained. In addition, M93 GAS was the most common from the population studied, which is consistent with a previous study of Thai GAS isolates. This PCR-RFLP protocol has the potential for the rapid screening of GAS emm types, and would be able to characterize the number of C-region in different types of GAS. This method is considerably advantageous as an alternative M typing approach for epidemiological studies in developing countries in which GAS is endemic and DNA sequencer and budgets are limited.