Conformation Polymorphism Analysis Research Articles

Analysis of Clinical and Biochemical Characteristics of Patients With Genetically Confirmed Familial Hypercholesterolemia in Russian North Western District Residents.

Aim To compare results of clinical, laboratory, and genetic examination of patients with familial hypercholesterolemia (FHC).Material and methods 112 patients aged 40.2±17.9 years (49 men) were examined. The gene of low-density lipoprotein receptor (LDLR) was analyzed and evaluated using the Dutch Lipid Clinic Network (DLCN) criterion of lipid score ≥6. The LDLR gene mutation was searched for using the conformational polymorphism analysis followed by sequencing of the DNA of isolated LDLR gene exons.Results Mean variables of the blood lipid profile were total cholesterol (C), 10.12±2.32 mmol/l, LDL-C, 7.72±2.3 mmol/l. Corneal arcus was observed in 15 % of patients, tendon xanthomas in 31.8 %, and xanthelasma palpebrarum in 5.3 %. The types of LDLR gene mutations included missense mutations (42.8 %), mutations causing a premature termination of protein synthesis (41.1 %), and frameshift mutations (16.1 %). In the presence of a mutation in exon 4, patients with IHD compared to patients with no IHD had significantly higher levels of total C (10.88±2.08 mmol/l vs. 8.74±1.57 mmol/l, respectively, р=0.001) and LDL-C (8.60±2.14 mmol/l vs. 6.62±1.79 mmol/l, respectively, р=0.005). Patients with IHD compared to patients with no IHD and a mutation in LDLR gene exon 9 had only a higher LDL-C level (8.96±1.53 mmol/l vs. 6.92±1.59 mmol/l, respectively, р=0.022). A differentiated comparison of IHD patients using a logistic regression depending on the identified type of LDLR gene mutation produced formulas for calculating the odds ratio of IHD and myocardial infarction (MI) with adjustments for the patient's age and baseline LDL.Conclusion The detection rate of the LDLR gene mutations was 42.8 % for missense mutations, 41.1 % for mutations causing a premature termination of protein synthesis, and 16.1 % for frameshift mutations. Blood lipid profiles did not differ between patients from different cities and with different types of LDLR gene mutations. Blood lipid profiles were different in IHD patients depending on the mutation type.

Polymorphism of bovine STAT5A gene and its association with milk production traits in crossbred cattle of Kerala, India

Signal transducer and activator of transcription 5A (STAT5A) is a transcription factor that mediates signals fromprolactin and growth hormone, the corresponding main regulators of lactation and growth of mammals. STAT5A issuggested to be candidate marker gene for quantitative traits in dairy cattle with respect to milk production traits. The objectives of present study were to investigate polymorphism of bovine STAT5A gene and its probable association with milk yield and milk-constituent traits in crossbred cattle. Crossbred cattle (250) in different farms of Kerala Veterinary and Animal Sciences University and different field centres of ICAR-Field progeny testing scheme,Thrissur were screened for polymorphism in exon-8 region of STAT5A gene using PCR-single-strand conformationpolymorphism analysis. A synonymous polymorphism (C/T) was found at position 1071 in exon 8. All three possiblegenotypes were identified with estimated frequencies of 0.584 (CC), 0.332 (CT) and 0.084 (TT); the estimated allelefrequencies were 0.75 (C) and 0.25 (T). The C/T genotypes demonstrated significant impact on milk fat per centbeing significantly higher in CC genotype cattle in comparison to other genotypes. It is evident by this investigationthat STAT5A polymorphism could be a promising indirect marker to improve milk production in crossbred cattle ofKerala.

Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae

Purpose: To compare the merits of conventional Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, and Direct-DNA sequencing in identification of drug resistance in M. leprae.
 Methods: A total of 41 leprosy cases were investigated for drug resistance using MFP assay, whereby growth results were obtained in 23 cases. The DNAs extracted from these samples were amplified in polymerase chain reaction (PCR) using specific primers in order to determine the drug resistance-loci. Furthermore, PCRSSCP analysis was carried out using PCR amplicons, while gel electrophoresis was performed to identify any shift in mobility in any one of the DNA strands, as well as the pattern of mutation.
 Results: A total of 5 isolates exhibited the highest degree of resistance R100, whereas 4 isolates showed intermediate level of resistance R10. In contrast, the least resistance was seen in R1. There were no mutations in 4 out of 10 strains which were dapsone-resistant, i.e., 1 HR, 2 IR and 1 LR. Moreover, there was no mutation in 305 bp sequence of the rpoB gene. DNA sequencing sensitivity was 60 %, whereas the sensitive isolates tested in the experiments exhibited no mutation, resulting in 100 % specificity.
 Conclusion: These results indicate the advantages of molecular techniques over conventional MFP technique. The study has revealed that PCR-SSCP analysis, a rapid, specific and less expensive method, has greater potential for use in routine testing of the susceptibility of M. leprae to rifampicin and dapsone, than the other tests.

Variation in the Ovine Glycogen Synthase Kinase 3 Beta-Interaction Protein Gene (GSKIP) Affects Carcass and Growth Traits in Romney Sheep.

Simple SummaryThe glycogen synthase kinase 3 beta (GSK3β)-interacting protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. To date, physiological function research into the GSK3β-interacting protein has been focused on cell lines, gene ‘knockout’ models, and over-expression studies, and to our knowledge, there have been no reports on how variation in the GSK3β-interacting protein gene (GSKIP) may affect phenotypic traits. In this study, PCR-SSCP methods were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two variant sequences were identified in exon 1 and this variation in GSKIP was associated with variation in lamb birth weight, hot carcass weight, and fat depth at the 12th rib.The glycogen synthase kinase 3 beta (GSK3β)-interacting protein (encoded by the gene GSKIP) is a small A-kinase anchoring protein, which complexes with GSK3βand protein kinase A (PKA) and acts synergistically with cAMP/PKA signaling to inhibit GSK3β activity. The protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. In this study, PCR-single strand conformation polymorphism (PCR-SSCP) analyses were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two SSCP banding patterns representing two different nucleotide variants (A and B) were detected in an exon 1 region, whereas in an exon 2 region only one pattern was detected. Variants A and B of exon 1 had one non-synonymous nucleotide difference c.37A/G (p.Met13Val). The birthweight of sheep of genotype AA (5.9 ± 0.06 kg) was different (p = 0.023) to sheep of genotype AB (5.7 ± 0.06 kg) and BB (5.7 ± 0.06 kg). The hot carcass weight (HCW) of sheep of genotype AA (17.2 ± 0.22 kg) was different (p = 0.012) to sheep of genotype AB (17.6 ± 0.22 kg) and BB (18.0 ± 0.29 kg), and the fat depth at the 12th rib (V-GR) of sheep of genotype AA (7.7 ± 0.31 mm) was different (p = 0.016) to sheep of genotype AB (8.3 ± 0.30 mm) and BB (8.5 ± 0.39 mm). The results suggest that the c.37A/G substitution in ovine GSKIP may affect sheep growth and carcass traits.

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.

Variation in the yak lipin-1 gene and its association with milk traits.

The aim of this research was to identify variation in the yak lipin-1 gene (LPIN1) and determine whether this variation affects milk traits. PCR-single stranded conformational polymorphism (PCR-SSCP) analysis was used to detect variation in the 5' untranslated region of LPIN1 in 500 yaks from four populations: Tianzhu white yaks, Qinghai yaks, wild × domestic-cross yaks and Gannan yaks. Four unique PCR-SSCP patterns, representing four different DNA sequence variants (named A, B, C and D), were observed. These contained six single nucleotide polymorphisms. Female Gannan yaks with BC genotype produced milk with a higher fat content (P < 0.001) and total milk solids (P < 0.001), than those with the AA, AB and BB genotypes. These results would suggest that LPIN1 is having an effect on yak milk fat synthesis.

Investigation of myostatin and calpain 3 gene polymorphisms and their association with milk-production traits in Sfakia sheep

Context Genetic selection based on genetic markers for economically important traits in Sfakia sheep. Aims The aim of the present study was to investigate variation in the ovine myostatin gene (MSTN) and calpain 3 gene (CAPN3), and their association with milk-production traits. Methods Records for milk yield, milk fat content, protein content, lactose content, and non-fat solid content, pH and somatic-cell score (log), were obtained from 376 Sfakia ewes. Polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) analyses were used to detect variation in intron 1 of MSTN and exon 10 of CAPN3. General linear models were then used to test for associations between the variation in MSTN and CAPN3, and milk-production traits. Key results The SSCP banding patterns for MSTN showed four variants (A1, A2, A3 and A4), which contained nine nucleotide sequence differences. Four SSCP banding patterns (C1, C2, C3 and C4) were observed for CAPN3 and these contained eight nucleotide-sequence differences. The MSTN variation was associated (P &amp;lt; 0.05) with variation in milk yield and non-fat milk solid content. Variation in CAPN3 was associated with milk yield (P &amp;lt; 0.001), fat content (P &amp;lt; 0.05) and lactose content (P &amp;lt; 0.05). Association analyses between the presence/absence of MSTN and CAPN3 variants and milk-production traits showed that a variant of MSTN that had previously between associated with muscle hypertrophy was associated with decreased milk yield (P &amp;lt; 0.05) and a lower non-fat milk solid content (P &amp;lt; 0.01). A CAPN3 variant that had previously been associated with increased sheep-carcass loin lean-meat yield was associated with a decreased milk yield (P &amp;lt; 0.01) and a decreased milk fat content (P &amp;lt; 0.05). Conclusions Our results have provided an insight into the effects of variation in ovine MSTN and CAPN3 on milk-production traits in sheep. Implications To preserve the dual-purpose characteristics of Sfakia sheep, breeding goals should take into account the possible antagonism between meat and milk traits.

"Mirror" Method to Estimate Mutagenic Activity of DNA Lesions.

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

High genetic stability of potato yellow mosaic Panama virus infecting tomato in Panama

The relevant regions in Panama involved in commercial tomato production, including the Chiriqui, Veraguas, Herrera, Los Santos and Panama Oeste provinces, were surveyed for the distribution and genetic diversity of potato yellow mosaic Panama virus (PYMPV) in the growing seasons of 2011 and 2012. A total of 28 tomato plots were surveyed and 314 individual tomato plants were sampled. DNA was extracted from each plant for a subsequent rolling circle amplification (RCA) analysis, to confirm the presence of begomovirus infections. The samples displaying a positive RCA reaction were subsequently analysed by PCR with a specific primer pair to identify PYMPV. This virus was detected in samples collected in all the above-mentioned provinces. The population structure of PYMPV was assessed by single-strand conformation polymorphism (SSCP) analysis. In total, four distinct SSCP patterns were distinguished. Based on the geographical distribution and the different patterns obtained, 42 viral isolates were selected for cloning and sequencing. Sequences obtained were compared with the ancestral sequence of PYMPV reported in Panama in 1998. These sequences showed very low variability, with two lineages probably originated by a single introduction.

Low frequency of p53 and k-ras codon 12 mutations in non-small cell lung carcinoma (NSCLC) tumors and surgical margins.

Lung cancer is one of the most common cancers and has became a predominant cause of cancer-related death throughout the world. The k-ras codon 12 mutation, which is the most common lung cancer mutation, is found in 15 to 30% of all lung cancers. Furthermore, the p53 gene has a very important role in the biological properties of tumor cells, and it is mutated in about 50% of non-small cell lung cancers. Residual tumor cells remain in surgical margins diagnosed as tumor free by histopathological techniques, and they can play a role in forming any local recurrence. Molecular methods may be exploited that are sensitive enough to detect small numbers of tumor cells. In the present study, we examined p53 gene mutations and k-ras codon 12 mutations from the tumor samples and surgical margins of 34 non-small-cell lung cancer patients. P53 gene mutations were analyzed by single strand conformational polymorphism analysis, heterodublex analysis and DNA sequencing. K-ras codon 12 mutations were analyzed by the mutagenic PCR-restricted fragment length polymorphism method. A p53 mutation was detected only in primary tumors of 3 out of 34 patients (8.82%). These mutations were clustered in exon 5. Moreover, a k-ras codon 12 mutation was detected in both the primary tumor and the surgical margin tissues of 2 out of 34 patients (5.88%). The detected mutation rate was low, in the range given in the literature. We think that different mechanisms related to other genes and individual genetic differences might play a role in cancer formation in our study group. We believe that molecular studies are necessary to identify biomarkers and to determine genetic alterations in histopathologically normal surgical margins.

Prevalence of the CYP2C19*2 (681 G>A), *3 (636 G>A) and *17 (‑806 C>T) alleles among an Iranian population of different ethnicities.

Polymorphisms in the cytochrome P (CYP) 450 family may cause adverse drug responses in individuals. Cytochrome P450 2C19 (CYP2C19) is a member of the CYP family, where the presence of the 681 G>A, 636 G>A and 806 C>T polymorphisms result in the CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles, respectively. In the current study, the frequency of the CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles in an Iranian population cohort of different ethnicities were examined and then compared with previously published frequencies within other populations. Allelic and genotypic frequencies of the CYP2C19 alleles (*2, *3 and *17) were detected using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis, PCR-single-strand conformation polymorphism analysis and DNA sequencing from blood samples of 1,229 unrelated healthy individuals from different ethnicities within the Iranian population. The CYP2C19 allele frequencies among the Iranian population were 21.4, 1.7, and 27.1% for the CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles, respectively. The frequency of the homozygous A/A variant of the CYP2C19*2 allele was significantly high and low in the Lur (P<0.001) and Caspian (P<0.001) ethnicities, respectively. However, the frequency of the homozygous A/A variant of the CYP2C19*3 allele was not detected in the Iranian cohort in the current study. The frequency of the heterozygous G/A variant of the CYP2C19*3 allele had the significantly highest and lowest frequency in the Fars (P<0.001) and Lur (P<0.001) groups, respectively. The allele frequency of the homozygous T/T variant of the CYP2C19*17 allele was significantly high in the Caspian (P<0.001) and low in the Kurd (P<0.05) groups. The frequency of the CYP2C19 alleles involved in drug metabolism, may improve the clinical understanding of the ethnic differences in drug responses, resulting in the advancement of the personalized medicine among the different ethnicities within the Iranian population.

Conformational chiral polymorphism in cis-bis-triphenylphosphine complexes of transition metals

Analysis of conformation polymorphism in cis-bis-triphenylphosphine complexes points to the importance of coordination numbers and homochirality of PPh3 ligands.

Analysis of virus structure variants in before- and after-thermotherapy-treated apple plants

ABSTRACTPre-thermotherapy apple plants and their homologous regenerated virus-infected apple plants were used to analyze the effect of high temperature on the structure of plant virus variants. The target bands of apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple mosaic virus (ApMV) in the plants of the two populations were purified and cloned, and 16 positive clones of each sample were randomly selected for single-strand conformation polymorphism (SSCP) analysis. The results showed that a predominant haplotype could be found in the two populations. The main variant was the same both in the regenerated and homologous treated plants, but the percentages in regenerated plants could be lower than, the same as, or higher than that in pre-thermotherapy plants. Two types could be divided depending on the percentage of the predominant haplotype in the treated plants, diversity (less 70%) and simple (more 80%), respectively. The clones of different haplotype in each isolates were sent to sequence, and the alignment results in accordance with that of the SSCP analysis. All the results indicated that thermotherapy could not affect the predominant variants of virus in treated plants, just change its percentage.

DEVELOPMENT AND EVALUATION OF A NOVEL PROBE MICROARRAY FOR GENOTYPING CITRUS TRISTEZA VIRUS USING AN INTEGRATED LAB-ON-CHIP DEVICE

An In-Check system was designed for Citrus tristeza virus (CTV) isolates genotyping. The system includes a set of 44 probes and enables a RT-PCR and microarray hybridization to be handled in a miniaturized silicon lab-on-chip (LoC) device. The 44 probes were designed based on 5'UTR, ORF1a, RdRp, p20, p23 and p33 genes. A probe on the p27 gene was used as a positive control. Capture probes were designed on variable regions of five phylogenetic groups corresponding to T36, T30, VT, NZ-RB, T68 CTV strains and the emerging HA16-5 and NZM16 isolates. Twenty-two isolates collected in Italy, inducing a range of symptoms spanning from vein clearing and slight to strong stem pitting on alemow, to seedling yellows of sour orange, were tested by the LoC device leading to the identification of T30, VT and T30+VT (mixed infections) groups. Eleven out of 23 probes specific for these genotypes reacted producing different patterns among and inside the groups. Results using the LoC device were comparable with capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis and multiple molecular marker (MMM) assays. The preliminary results demonstrate that the device is highly robust and fast.

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.

Novel single nucleotide polymorphisms in the 5' regulatory region of the duck SCD1 gene and their associations with serum biochemical levels and fatty acid composition.

Stearoyl-coenzyme A desaturase 1 (SCD1) is the key limiting enzyme in the synthesis of monounsaturated fatty acids, and plays a crucial role in the regulation of oleic acid. In this study, 165 ten-week-old Cherry Valley ducks were used to investigate single nucleotide polymorphisms (SNPs) in the 5' regulatory region of the SCD1 gene, and their associations with duck serum biochemical levels and fatty acid composition. Two novel SNPs, g.936516 C > G and g.936551 T > C, were found by polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing methods, exhibiting six genotypes (AA, BB, CC, AB, AC, and BC). The frequency of the dominant genotype BB and allele B was 0.321 and 0.403, respectively. The polymorphism information content value was 0.617, indicating high polymorphism. The chi-square test indicated that the genotype distribution deviated markedly from Hardy-Weinberg equilibrium (P < 0.01). The linkage of the two mutant sites in the duck SCD1 gene had significant effects on the serum albumin, total protein, globulin, triglyceride, total cholesterol, and cholinesterase levels, as well as on 16 kinds of fatty acids except for C14:1 and C20:0 (P < 0.05). These results indicated that the C allele might have a positive effect on polyunsaturated fatty acids with potential health benefits. Therefore, the SCD1-gene-specific SNPs in the 5' regulatory region may be a useful marker for serum lipid, serum protein, and fatty acid composition in future marker-assisted selection for duck breeding.

Characterisation of exon 9 of solute carrier family 11 member A1 gene in Vechur cattle

The solute carrier family 11 member A1 (SLC11A1) gene has been associated with natural resistance to intracellular pathogens such as Brucella, Salmonella, Leishmania and Mycobacterium in several species including bovine and plays a critical role in elimination of pathogens by generating hydroxyl free radicals. The objective of the present study was to investigate the polymorphism in exon 9 of SLC11A1 gene in Vechur cattle, one of the dwarf cattle of India which is known for its disease resistance. A 198 bp fragment containing exon 9 of the gene was amplified by polymerase chain reaction (PCR). The amplicons upon single strand conformation polymorphism (SSCP) analysis revealed two different banding patterns. A novel non synonymous SNP (g.46C&gt;T) with predominance of CC genotype was also detected in Vechur cattle. These results suggest that there exists a considerable genetic variation at SLC11A1 locus and further association studies may help in development of a PCR based genotyping assay to select cattle with better immunity to intracellular pathogens.

In vitro activity of rifampin against and rpoB mutations in Chlamydia trachomatis clinical isolates

Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them. Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates. The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP) analysis. Sequencing of PCR products was carried out for two clinical isolates. Results No rifampin-resistant strain was found among these clinical isolates. The MIC of rifampin varied from 0.004 to 0.030 mg/L. Neither SSCP analysis nor sequencing showed rpoB mutations. Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin. The unresponsiveness to rifampin may be attributed to multiple factors. Key words: Chlamydia trachomatis; Rifampin; Microbial sensitivity tests; Drug resistance, microbial; DNA mutational analysis; Genes, rpoB

Polymorphism of polygalacturonase-inhibiting protein genes by fluorescence-based PCR single-strand conformation profiling and sequencing in autotetraploid alfalfa (Medicago sativa) populations

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins involved in plant defence. The aim of the current study was to isolate the genomic fragment of PGIP genes in alfalfa (Medicago sativa) and to analyse their sequence variation. Four PGIP genes, namely MsPGIP1, MsPGIP2, MsPGIP3 and MsPGIP4, were discovered using the homology cloning method, and sequence variation among these four PGIP genes was higher than those among PGIP genes reported in non-Medicago leguminous plants. Fluorescence-based PCR single-strand conformation polymorphism (FPCR–SSCP) analysis, which was further improved by adding primers to PCR products, combined with sequencing revealed that MsPGIP4 had the most alleles (17) and most amino acid variation sites (12) while MsPGIP1 had the least (8 and 3, respectively). Furthermore, the extents of allele frequency divergence in MsPGIP1 and MsPGIP4 were obviously greater than those of MsPGIP2 and MsPGIP3 among the studied populations. Three populations, namely ‘Rambler’, ‘C/W5’ and ‘PG sutter’, showed higher frequency divergence for three of the four PGIP genes than other studied populations. In conclusion, the four alfalfa PGIP genes found in this study possessed high intergene and intragene sequence variation, and showed different extents of allele frequency divergence among the studied eight alfalfa populations. Overall, this study lays a foundation for studying their biological and molecular functions. In addition, the improved SSCP analysis technique described here provides a useful tool for molecular biology research in autopolyploids.

CORRELATION ANALYSIS BETWEEN MYOSTATIN GENE POLYMORPHISMS AND CARCASS TRAITS IN NEW ZEALAND ROMNEY SHEEP

Using genetic markers can aid identifying those animals with the highest values for economically important traits in sheep. The current study was designed to detect the allelic and genotypic polymorphisms of the intron 1 of myostatin gene and to test their associations with carcass traits (slaughtering weight, dressing%, shoulder yield, loin yield, leg yield, total yield, shoulder yield%, loin yield% and leg yield%) in 529 male lambs of New Zealand Romney sheep. The polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to identify the allelic and genotypic polymorphisms in intron 1 of myostatin gene for 529 males from New Zealand Romney lambs. Associations of the variation in the intron 1 of myostatin gene with carcass traits were determined using the general linear model (GLM) procedure of SAS (2000). The SSCP analysis revealed six SSCP genotypes: AA, AB, AC, BB, BC and CC with frequencies of 0.107, 0.368, 0.100, 0.289, 0.129 and 0.005, respectively, that derived from three identified alleles: A, B and C with frequency 0.34, 0.54 and 0.12, respectively. Myostatin genotype significantly affected (P˂0.05) slaughtering weight and total yield, and highly significant affected (P˂0.001) dressing%, leg yield, shoulder yield%, loin yield% and leg yield%. The presence of A allele in animal genotype was associated with higher leg yield and leg yield%, however, the presence of B allele was associated with higher loin yield and loin yield%. The LSM showed that, lambs with two copies of A allele had the highest dressing%, leg yield, leg yield% and total yield, however, lambs with two copies of B allele had the highest shoulder yield, shoulder yield% and loin yield%. The results presented here give valuable information to select for A and B alleles and against C allele to improve the most important primal cuts of lambs across most production systems.