Single-strand Conformation Polymorphism Band Research Articles

Genetic variation in the ovine KAP22-1 gene and its effect on wool traits in Egyptian sheep.

The objective of this study was to investigate the genetic polymorphisms in the keratin-associated protein (KAP22-1) gene in Barki (), Rahmani () and Ossimi () as the three major sheep breeds in Egypt. Subsequently, the detected variants were correlated with important wool traits. The traits included greasy fleece weight (GFW, g), staple length (SL, cm), prickle factor (PF, %), medullated fiber (MF, %), fiber diameter (FD, m), crimp percentage (CR, %) and the standard deviation of FD (SD, m), as well as the subjectively assessed traits of kemp score (KS), handle grade (HG), greasy color grade (GCG), bulk grade (BG), luster grade (LG) and staple structure (SST). Animals were genotyped by polymerase chain reaction (PCR) – single strand conformation polymorphism (SSCP). Five SSCP banding patterns representing three different nucleotide variants (A, B and C) were detected. DNA sequencing confirmed three single nucleotide polymorphisms (SNPs). Animal age significantly affected GFW (), SD (), SL (), CR (), KS (), LG () and SST (). Likewise, the breed had a significant effect on all studied traits except HG and BG, which was not significant. Results showed significant associations between the KAP22-1 variants and CR (), SL (), KS () and GCG (). Interestingly, animals with BB genotypes tended to produce more wool yield ( g) with high SL ( cm), CR ( %) and KS (). Results of this study strongly recommend the KAP22-1 gene as a candidate gene for wool production traits in Egyptian sheep, with new useful insights into the visually assessed wool traits. The identified genetic markers may be incorporated into breeding strategies and genetic improvement programs of wool traits in Egyptian sheep.

NOVEL ASSOCIATION BETWEEN GnRHR GENE AND GROWTH TRAITS IN AWASSI AND KARAKUL SHEEP

This study was conducted to investigate the association of the Gonadotropin-Releasing Hormone Receptor (GnRHR) gene with growth traits in Awassi (n= 123) and Karakul (n= 78) sheep. Starting from the birth-day to 12 months of age, several growth traits were investigated, namely body weight and length, wither and rump height, chest, and abdominal circumferences. Four fragments were designed to amplify three exons of GnRHR gene, P1 and P2 for exon 1, P3 for exon 2, and P4 for exon 3. The polymorphisms in these amplicons were analyzed by single-strand conformation polymorphism (SSCP) method. Genotypic and allele frequency of GnRHR gene were computed after sequence alignment. The results detected two different SSCP banding patterns GG and GA for both P2 and P3 amplicons, and two novel mutations were observed in GA genotype of P2 and P3 amplicons respectively, p.N87= and p.L242F. The identified p.N87= exhibited no relationship with all measured growth traits, while p.L242F showed significant association with the body weight, length, and abdominal circumference in month-6 and month-9. This association was increasingly observed in month-12 with all growth traits, in which individuals with GA genotype exhibited higher growth traits measurements than individuals with GG genotype (P < 0.01). In conclusion, the detected polymorphism of GnRHR gene exon-2 may be useful in marker-assisted selection for growth traits in Awassi and Karakul breeds. This study is the first one to describe the effect of GnRHR polymorphisms on growth traits, which confirm the potential usefulness of this gene in marker-assisted selection in sheep.

NOVEL ASSOCIATION BETWEEN GnRHR GENE AND GROWTH TRAITS IN AWASSI AND KARAKUL SHEEP: GnRHR genetic polymorphism in sheep

This study was conducted to investigate the association of the Gonadotropin-Releasing Hormone Receptor (GnRHR) gene with growth traits in Awassi (n= 123) and Karakul (n= 78) sheep. Starting from the birth-day to 12 months of age, several growth traits were investigated, namely body weight and length, wither and rump height, chest, and abdominal circumferences. Four fragments were designed to amplify three exons of GnRHR gene, P1 and P2 for exon 1, P3 for exon 2, and P4 for exon 3. The polymorphisms in these amplicons were analyzed by single-strand conformation polymorphism (SSCP) method. Genotypic and allele frequency of GnRHR gene were computed after sequence alignment. The results detected two different SSCP banding patterns GG and GA for both P2 and P3 amplicons, and two novel mutations were observed in GA genotype of P2 and P3 amplicons respectively, p.N87= and p.L242F. The identified p.N87= exhibited no relationship with all measured growth traits, while p.L242F showed significant association with the body weight, length, and abdominal circumference in month-6 and month-9. This association was increasingly observed in month-12 with all growth traits, in which individuals with GA genotype exhibited higher growth traits measurements than individuals with GG genotype (P < 0.01). In conclusion, the detected polymorphism of GnRHR gene exon-2 may be useful in marker-assisted selection for growth traits in Awassi and Karakul breeds. This study is the first one to describe the effect of GnRHR polymorphisms on growth traits, which confirm the potential usefulness of this gene in marker-assisted selection in sheep.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

Association of Matrilin-3 Gene Polymorphism with Temporomandibular Joint Internal Derangement

Temporomandibular joint internal derangement (TMJ ID) is a multifactorial complex disease characterised by articular disc degeneration. Matrilin-3 is a cartilage and bone-specific adaptor protein, and amino-acid substitutions in the protein are associated with skeletal diseases and joint disorders. We aimed to detect the variants of Matrilin-3 gene (MATN3) in a TMJ ID case-control group and to investigate the risk association of the detected variants with TMJ ID. A case control study was conducted consisting of 57 unrelated TMJ ID patients (32.7 ± 8.2) and 96 unrelated healthy controls (26.63 ± 3.05) without TMJ ID to look for associations with variants of the MATN3 gene. DNA from individual subjects was extracted and each of the eight exons was amplified by polymerase chain reaction using and analyzed by single-strand conformational polymorphism (SSCP) analysis. SSCP variants were subjected to DNA sequence analysis, which yielded band pattern variations in exon 2 of the gene. We further analyzed exon 2 by DNA sequencing to determine the sequence of these variants. We identified SSCP band patterns variants in exon 2 of the MATN3 gene which upon sequencing revealed a single C to T transition mutation (rs28598872) c.447 C>T (g.11608 C>T). This polymorphism is predicted to result in a synonymous mutation (pAla149 = ). The TT and CT genotypes were more prevalent than the CC genotype in TMJ ID patients compared to the control group with a risk factor of 2.12 (confidence intervals [CI] :0.88-5.08) and 2.0 (CI:0.726-5.508). In addition, TMJ ID patients were divided into two groups as anterior disc displacement with reduction (ADDWR) and anterior disc displacement without reduction (ADDWOR) and compared with the controls. The TT and CT genotypes were more prevalent than the CC genotype in ADDWR patients compared to the control group with a risk factor of 3.85 (CI:0.927-16.048) and 3.75 (1.02-13.786), respectively. We found that, among ADDWR patients, the T allele is a risk factor both in homozygous and heterozygous carriers (p < 0.052, p < 0.036). The results of the study indicate a potential role for the MATN3 rs28598872 polymorphism in the pathogenesis of TMJ ID.

Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5′-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

Culture -independent Pathogenic Bacterial Communities in Bottled Mineral Water

Bottled mineral water (BMW) is an alternative to mains water and consider it to be better and safer. Access to safe BMW from the bacteria involving potential health hazard is essential to health. Cultivation-independent technique PCR-based single-strand conformation polymorphism (SSCP) for genetic profiling of PCR-amplified 16S rRNA genes was performed using Com primer set targeting the 16S rRNA genes for detection of pathogenic bacteria in bottled mineral water from the final product of six factories for bottled mineral drinking water in Wadi El-natron region- Egypt. These factories use often ozone technology to treat large quantities of water because of its effectiveness in purifying and conditioning water. A total of 27 single products were isolated from the profiles by PCR re-amplification and cloning. Sequence analysis of 27 SSCP bands revealed that the 16S rRNA sequences were clustered into seven operational taxonomic units (OTUs) and the compositions of the communities of the six samples were all common. The results showed that most communities from phyla Alphaproteobacteria and certainly in the Sphingomonas sp . Culture-independent approaches produced complementary information, thus generating a more accurate view for the bacterial community in the BMW, particularly in the disinfection step, as it constitutes the final barrier before BMW distribution to the consumer

Genetic Variability of Hepatitis C Virus (HCV) 5' Untranslated Region in HIV/HCV Coinfected Patients Treated with Pegylated Interferon and Ribavirin.

Association between hepatitis C virus (HCV) quasispecies and treatment outcome among patients with chronic hepatitis C has been the subject of many studies. However, these studies focused mainly on viral variable regions (E1 and E2) and usually did not include human immunodeficiency virus (HIV)-positive patients. The aim of the present study was to analyze heterogeneity of the 5'untranslated region (5'UTR) in HCV/HIV coinfected patients treated with interferon and ribavirin. The HCV 5’UTR was amplified from serum and peripheral blood mononuclear cells (PBMC) samples in 37 HCV/HIV coinfected patients treated for chronic hepatitis C. Samples were collected right before treatment, and at 2, 4, 6, 8, 12, 20, 24, 36, 44, 48, 60, and 72 weeks. Heterogeneity of the 5'UTR was analyzed by single strand conformational polymorphism (SSCP), cloning and sequencing. Sustained virological response (SVR) was achieved in 46% of analyzed HCV/HIV co-infected patients. Stable SSCP band pattern was observed in 22 patients (62.9%) and SVR rate among these patients was 23%. Decline in the number of bands and/or shift in band positions were found in 6 patients (17.1%), 5 (83%) of whom achieved SVR (p=0.009). A novel viral genotype was identified in all but one of these patients. In 5 of these 6 patients a new genotype was dominant. 5'UTR heterogeneity may correlate with interferon and ribavirin treatment outcome. In the analyzed group of HCV/HIV coinfected patients, viral quasispecies stability during treatment favored viral persistence, whereas decrease in the number of variants and/or emergence of new variants was associated with SVR. Among injection drug users (IDU) patients, a new genotype may become dominant during treatment, probably due to the presence of mixed infections with various strains, which have different susceptibility to treatment.

Hepatitis C virus 5′ untranslated region variability correlates with treatment outcome

Hepatitis C virus (HCV) variability affects viral-host interactions. We analysed HCV 5'untranslated region (5'UTR) in sera and peripheral blood mononuclear cells (PBMC) from chronic hepatitis C patients undergoing antiviral treatment. We studied 139 patients treated with pegylated interferon and ribavirin. The primary endpoint was a sustained virological response (SVR) defined as negative HCV RNA level 24 weeks after the end of therapy. 5'UTR was analysed by single-strand conformational polymorphism (SSCP) and sequencing. The pretreatment SSCP pattern in serum and PBMC differed in 26 (18.7%) patients. During therapy, the SSCP pattern remained stable in 65 (60.8%) patients, number of bands declined in 16 (15.0%), and in 18 (16.8%) patients, changes were qualified as 'shift' indicating change in band positions. In univariate analysis, there was a significant (P ≤ 0.05) positive association between SVR and pretreatment serum and PBMC dissimilarities, initial viral load <10(6) IU/mL, IL-28B CC genotype of the rs12979860 single nucleotide polymorphism and change in the SSCP band pattern (either 'shift' or decline) In multivariable analysis, only low initial viral load, IL-28B genotype, and changes in the SSCP band pattern were independent factors associated with SVR. In conclusion, stability of 5'UTR correlated with infection persistence, while changes correlated with SVR.

Hepatitis G virus/GBV-C in serum, peripheral blood mononuclear cells and bone marrow in patients with hematological malignancies

BackgroundHGV/GBV-C is highly prevalent in the general population but its significance remains unclear. It is known that HGV/GBV-C is not primary hepatotropic and its replication was reported in PBMC, bone marrow and other tissues. To investigate a possible role of HGV/GBV-C 115 consecutive patients with hematological malignancies were analyzed for virus RNA presence and quasispecies composition in three compartments: serum, PBMC and bone marrow. MethodsRT-PCR was used to amplify 5′UTR HGV/GBV-C in serum, PBMC and bone marrow. Viral sequences obtained from three compartments were subjected for comparative molecular analysis performed by single strand conformational polymorphism (SSCP) and pyrosequencing. ResultsHGV/GBV-C RNA was detected in 23 out of 115 (20.0%) patients, most often in bone marrow (18 patients), followed by PBMC (11 patients) and serum (10 patients). Differences in SSCP bands distribution corresponding to different viral variants and confirmed by direct sequencing were observed in three patients. ConclusionHGV/GBV-C infection is frequent in patients with hematological malignancies. Common detection of HGV/GBV-C in bone marrow supports the hypothesis that it is a major replication site of this virus.

Variability of hepatitis C virus hypervariable region 1 (HVR-1) during the early phase of pegylated interferon and ribavirin therapy

Genetic variability of hepatitis C virus (HCV) is considered to be an important factor defining viral pathogenesis, persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR-1) before and during the early period of pegylated interferon alfa (PEG-IFN-α) and ribavirin treatment in correlation with treatment outcome. The study involved 24 patients treated with PEG-IFN-α and ribavirin whose sera were collected before (baseline) and at 7, 14, 21 28 and 56 day of treatment. HCV HVR-1 region was amplified by nested RT- PCR and subjected to SSCP (single strand conformational polymorphism) analysis. SSCP changes of HCV HVR-1 over time in each patient were compared to treatment outcome results. In 2/11 (18%) SVR+ and 8/13 (62%) SVR- treated patients, HVR-1 genetic changes manifested by new SSCP bands (new genetic variants) and were significantly more frequent in nonresponders (P <0.05). Our results indicate that HCV HVR-1 variability during the early phase of PEG-IFN-α and ribavirin therapy may be predictive of treatment outcome.

Allelic polymorphism of Makoei sheep leptin gene identified by polymerase chain reaction and single strand conformation polymorphism

Leptin, a 16-kDa protein secreted from white adipocytes has been implicated in the regulation of food intake, energy expenditure and whole-body energy balance in animals. In the present study, the polymorphism of the leptin gene (LEP) of Makoei sheep was investigated by polymerase chain reaction and single strand conformation polymorphism technique (PCR–SSCP). Genomic DNA was extracted from whole blood samples collected from 130 sheep. A 471 bp LEP exon 3 segment was amplified by standard PCR, using the locus specific primers. PCR products were subjected to SSCP denaturation and polyacrylamide gel electrophoresis. SSCP bands were visualized with silver staining. Five SSCP patterns, representing five different genotypes, were identified. The frequencies of the observed genotypes were 0.17, 0.09, 0.14, 0.37 and 0.23 for AB, BB, AC, BC and CC, respectively. Allele frequencies were 0.15, 0.37 and 0.48 for A, B and C, respectively. The observed heterozygosity (H obs ) value for LEP gene was 0.6769. The chi-square test showed that there was no significant deviation (P>0.01) from Hardy-Weinberg equilibrium for this locus in Makoei sheep population. Key words: Leptin gene (LEP), polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) Makoei sheep.

Asymmetric single‐strand conformation polymorphism: An accurate and cost‐effective method to amplify and sequence allelic variants

An efficient alternative strategy to conventional cloning was needed to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies. This method would facilitate studies and minimize technical problems typically encountered in cloning methodologies. We tested a variety of single-strand conformation polymorphism (SSCP) protocols including purified and unpurified symmetric and asymmetric PCR, loading buffers, and electrophoresis conditions (buffers, matrix, running time, temperature). Results obtained from direct SSCP band sequencing were compared to those obtained from cloning. Our optimized protocol uses asymmetric PCR, with the majority of the samples run in polyacrylamide gel electrophoresis (PAGE). It consistently separated PCR products from 450 to 1200 bp. Asymmetric PCR single-strand conformation polymorphism is an efficient alternative technique for isolating allelic variants of highly heterozygous individuals, with its greatest applications in sequencing allopolyploids. It eliminates two common problems encountered in cloning: PCR recombination and heteroduplex fixation. In addition, our protocol greatly lowers costs and time associated with procedures.

Analysis of bacterial core communities in the central Baltic by comparative RNA–DNA-based fingerprinting provides links to structure–function relationships

Understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. To this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central Baltic Sea at four stations, which were up to 450 km apart and at a depth profile representative for the central part (Gotland Deep, 235 m). Bacterial community structure was followed by 16S ribosomal RNA (rRNA)- and 16S rRNA gene-based fingerprints using single-strand conformation polymorphism (SSCP) electrophoresis. Species composition was determined by sequence analysis of SSCP bands. High similarities of the bacterioplankton communities across several hundred kilometers were observed in the surface water using RNA- and DNA-based fingerprints. In these surface communities, the RNA- and DNA-based fingerprints resulted in very different pattern, presumably indicating large difference between the active members of the community as represented by RNA-based fingerprints and the present members represented by the DNA-based fingerprints. This large discrepancy changed gradually over depth, resulting in highly similar RNA- and DNA-based fingerprints in the anoxic part of the water column below 130 m depth. A conceivable mechanism explaining this high similarity could be the reduced oxidative stress in the anoxic zone. The stable communities on the surface and in the anoxic zone indicate the strong influence of the hydrography on the bacterioplankton community structure. Comparative analysis of RNA- and DNA-based community structure provided criteria for the identification of the core community, its key members and their links to biogeochemical functions.

Mutational Analysis of ASPP1 and ASPP2 Genes, a p53-related Gene, in Gastric and Cololorectal Cancers with Microsatellite Instability

To the editor, In most human cancers, functions of p53 tumor suppressor are inactivated by many mechanisms, most commonly by somatic mutation.1 Alternatively, p53 is inactivated by genes whose protein products interact with p53.1 Apoptosis-stimulating protein of p53-1 (ASPP1) and -2 (ASPP2) promote p53-mediated apoptosis by enhancing its transactivation function.2 ASPP2+/- mice suffer from an increased incidence of tumorsm,3 suggesting it as a haploinsufficient tumor suppressor gene. However, neither of ASPP1 or ASPP2 mutation has been reported in cancers. Microsatellite instability (MSI) is characterized by length alterations in repeated DNA sequences, and 10-30% of colorectal cancer (CRC) and gastric cancer (GC) are categorized as MSI-positive cancers.4 In a public database, we found an A7 repeat in exon 8 of ASPP1 and an A7 repeat in exon 6 of ASPP2. To see whether these A7 are mutated in GC and CRC with MSI, we analyzed them by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) as described previously.5 We analyzed 32 high-MSI (MSI-H) and 13 low-MSI (MSI-L) GC, and 38 MSI-H and 15 MSI-L CRC. We used two primer pairs that amplified each A7 of ASPP1 and the ASPP2. Tumor and normal DNA from microdisscted cells were amplified by the PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. The PCR-SSCP analysis of ASPP2 identified aberrant bands in one GC with MSI-H (3.1%) and two CRC with MSI-H (5.3%), but none in those with MSI-L (Fig. 1). Normal DNA of the patients showed no shifts, indicating the mutations had risen somatically. Direct DNA sequencing identified an identical ASPP2 deletion mutation in the A7 repeat (c.576delA), which would result in a premature stop (p.Val193fsX1) and resembled a typical loss-of-function mutation. For ASPP1, there was no aberrantly migrating SSCP band in the cancer tissues. Fig. 1 Mutation of ASPP2 in a gastric carcinoma with high-microsatellite instability. (A) PCR product of ASPP2 from a colon carcinoma shows aberrant bands (arrows in lane T) as compared to single-strand conformation polymorphism from normal tissue (lane N) of ... As a possible mechanism for p53 inactivation, we analyzed ASPP1 and ASPP2 mutations in their repeat sequences and found some of the cancers with MSI-H harbored ASPP2 frameshift mutation. In colon, tumors with MSI have frameshift mutations in specific target genes, and fewer mutations are found in K-RAS and p53.4 Although the incidence of the ASPP2 mutation is not high, our data indicate that the ASPP2 mutation could partially explain a p53-inactivating mechanism in GC and CRC with MSI. Four ankylin repeats (amino acids 930-1047) and an SH3 domain (amino acids 1064-1114) of ASPP2 are essential for the interaction of ASPP2 with p53.2,3,6 The frameshift mutation identified in this study (p.Val193fsX1) would remove ankylin repeats and SH3 domain, and pro-apoptotic function of ASPP2 may be decreased in the affected cancers. Our data suggest that decreased p53 functions by the ASPP2 mutation might possibly contribute to pathogenesis in MSI-H cancers. Whether inactivation of ASPP genes further contributes to GC and CRC development in addition to the frameshift mutation of ASPP2, mutational analysis of entire coding region of ASPP1 and ASPP2 genes is required in future studies.

Fungal composition of lichen thalli assessed by single strand conformation polymorphism

AbstractFungi that are unrelated to the mycobiont species frequently colonize lichens. Some of these fungal colonists are described lichenicolous fungi, lichen parasites and pathogens that produce recognizable morphological characters, while others apparently produce no noticeable structures. Here we apply the single strand conformation polymorphism (SSCP) technique to directly assess the abundance of different fungi in lichens. Twenty-eight lichen thalli were chosen, some with and some without externally visible symptoms of parasite infection, and these were subjected to total DNA extraction. PCR was conducted with fungal-specific primers for the ITS region of ribosomal DNA. Single strands of the products were separated on native acrylamide gels. The majority of lichen specimens, both infected and those without symptoms, displayed more than one band in the stained gels. In one case, 14 bands were detected using SSCP. Some of these bands apparently represent other neighbouring lichens in the habitat, but many are apparently non-lichen-forming. Since few lichen-associated fungi have been cultured and sequenced, it is difficult to know if SSCP bands represent obligate lichenicolous fungi, other asymptomatic lichen parasites, or fungi not obligately associated with lichens, but our results indicate that large numbers of non-lichen-forming fungi commonly co-occur with lichens in nature. For specimens of the filamentous lichensCystocoleus ebeneusandRacodium rupestrewe used cloned sequences to compare the number of sequences obtained by the SSCP method to the number obtained by direct sequencing of thallus extracts, and we generally found that more sequences could be detected by SSCP than could be seen by direct sequencing.

Effect of the corn silage to grass silage ratio and feed particle size of diets for ruminants on the ruminal Bacteroides-Prevotella community in vitro

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1 mm or 4 mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of the feed particle size on this bacterial group.

Mutational Analysis of MITOSTATIN, a Candidate Tumor-Suppressor Gene, at a Mononucleotide Repeat in Gastric and Colorectal Carcinoma

To the editor, Human MITOSTATIN gene encodes a mitochondrial protein, which promotes apoptosis and inhibits cell growth.1 The MITOSTATIN gene is somatically mutated in cancer cell lines (gastric, prostate and pancreas cell lines) and is down-regulated in bladder and breast cancer tissues.1 These data suggest that MITOSTATIN gene may be a tumor suppressor gene, inactivation of which plays an important role in cancer development. Although somatic mutations of MITOSTATIN gene have been identified in cancer cell lines, its mutation status has not yet been studied in cancer tissues. Frameshift mutations of genes containing mononucleotide repeats are features of gastric (GC) and colorectal carcinomas (CRC) with microsatellite instability (MSI).2 Many cancer-associated genes, such as TGFBR2, BAX, IGFR2 and TCF4, have been found to harbor mutations at mono- or dinucleotide repeats in the coding sequences in these cancers with MSI.2 There is a polyadenine tract (A8) in the exon 5 of the MITOSTATINgene, but the frameshift mutation at this site is not known. To see whether the mononucleotide repeat A8 in the exon 5 of MITOSTATIN gene is mutated in GC and CRC, we analyzed the exon 5 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. For the mutation analysis, methacarn-fixed tissues of 42 GC and 51 CRC with MSI were used in this study. These cancers consisted of 31 GC with high MSI (MSI-H), 11 GC with low MSI (MSI-L), 37 CRC with MSI-H and 14 CRC with MSI-L. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator.3 Genomic DNA each from tumor cells and corresponding normal cells were amplified with a primer pair by PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. After SSCP, migration of the PCR products on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. Other procedures of the PCR and SSCP were described in our previous studies.3,4 On the SSCP, all of the PCR products from the cancers were clearly seen. The PCR-SSCP analysis identified aberrant bands in one (3.2%) of the 31 GC with MSI-H, but not in the other cancers. DNA from normal tissues from the same patients showed no evidence of the mutation in SSCP, indicating the mutations had risen somatically. A direct DNA sequence analysis of the GC with the aberrant SSCP bands led to identification of a MITOSTATIN deletion mutation within the A8 repeat sequences (Fig. 1). The mutation was c.522delA, which would result in premature stops of the amino acid synthesis (p.Lys174AsnfsX26). We repeated the experiments twice, including PCR, SSCP and DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent. Fig. 1 Frameshift mutation of MITOSTATIN in a gastric carcinoma with microsatellite instability. Direct DNA sequence analysis of MITOSTATIN exon 5 from a gastric adenocarcinoma shows a heterozygous A deletion in tumor tissue relative to normal tissue. Failure of apoptosis allows survival of cells and plays an important role in development of cancers.3 As a mechanism of apoptosis resistance in cancer cells, somatic mutations of pro-apoptosis genes have been reported in many human cancers, and many of the mutations were proven to inactivate cell death.3,5 Because the main tumor suppressor function of MITOSTATIN appears to depend on apoptosis activity, we analyzed frameshift mutation of MITOSTATIN gene at a mononucleotide repeat that may inactivate the apoptosis. However, we detected only one MITOSTATIN mutation in the cancers with MSI, indicating that the mutation at the repeat is rare in GC and CRC with MSI. In the earlier study1 that first disclosed tumor suppressor functions of MITOSTATIN, the authors found that loss of expression and mutation of the gene in solid tumors and cell lines, respectively. Our and the earlier data1 suggest that MITOSTATIN may be inactivated by various mechanisms. To see whether loss of function of MITOSTATIN is a feature of human cancers, the status of MITOSTATIN gene should be further analyzed at various levels in many cancer types.

Quasispecies of genotype 4 of hepatitis C virus genomes in Saudi patients managed with interferon alfa and ribavirin therapy.

BACKGROUND AND OBJECTIVES:Many patients with hepatitis C virus (HCV) infection do not respond to antiviral treatment, possibly due to viral quasispecies. We aimed to investigate whether the quasispeices population could be used as a predictor of response to therapy in our patients.METHODS:The quasispecies of HCV genotype 4 (HCV-4) were studied in 25 naïve Saudi patients at zero, three, and six months following interferon alfa and ribavirin combination therapy. Hypervariable region 1 within the E2/NS1 gene of the virus was analyzed by the single-strand conformation polymorphism (SSCP) technique after amplification.RESULTS:Pretreatment DNA bands by SSCP (2-7 bands) were detected in all patients. In those who achieved a complete virological response within six months (viral load <0.2 Meq/mL; n=7), bands ranged from 2-6 (mean = 3.71±1.25). In six of these seven patients, the number of SSCP bands remained either the same or decreased sequentially. In those patients who did not respond (viral load >0.2 Meq/mL; n=18), the bands also ranged from 2-7; mean 3.77±1.73. In six of these non-responding patients, the SSCP bands remained the same or decreased sequentially. There was no significant difference between pretreatment quasispecies composition and response (P=·53). Two of the four patients with pretreatment high viral load and the same or decreased composition of quasispecies bands responded to the therapy.CONCLUSION:Quasispecies in our studied patients cannot be used to predict responsiveness to treatment, but may offer an explanation for failure of most HCV-4 patients to respond to interferon alfa and ribavirin therapy.

Distribution of prokaryotic organisms in a tropical estuary influenced by sugar cane agriculture in northeast Brazil

In a joint Brazilian-German case study, distribution patterns of microorganisms were compared with environmental variables in the tropical coastal Manguaba lagoon in northeast Brazil, which is situated downstream of several sugar cane processing plants . 16S rDNA and 16S rRNA single strand conformation polymorphism (SSCP) gene fingerprinting were used to follow the composition and distribution of microorganisms throughout the salinity gradient of the lagoon. Potentially abundant microorganisms were identified by sequencing representative SSCP bands. It could be demonstrated that the distribution of microbes was in close relation to the physico-chemical environmental settings and followed a common scheme. In the in- and outlet areas of the lagoon rather transient microbial communities were found, whereas in the central part a stable, diverse community was encountered, that due to the long residence time of the water, had ample time for development and adaptation.