Absence Of Restriction Site Research Articles

Biochemistry Characterization of Type 2 Diabetes in Tunisia and their Relationship with 4545G/C Polymorphism Located in Adiponectin Gene

Diabetes incidence is increasing worldwide. Many studies demonstrated that polymorphisms within the adiponectin gene could be associated with type 2 diabetes mellitus (T2DM). A case–control study was conducted to find the association between SNP 4545G/C and T2DM in Tunisian population. The study included 561 patients referring to diabetic and 437 healthy controls. DNA was extracted from blood and genotyped by PCR–RFLP by using (HinfI) enzyme. Prevalence of obesity (p <0.001), dyslipidemia (p <0.001), hypertension (p <0.001) were significantly increased in cases with report to witnesses. Also for the lipid parameters Urea, CRP and HDL-C were significantly (p <0.001) and HDL / LDL (p = 0.983) are decreased in diabetics compared to controls two types of alleles G (Absence of restriction site) and C (presence of the restriction site) respectively of frequencies (0.76 and 0.23) in the controls and (0.73 and 0.26) in the diabetics, as well as three genotypes GG, GC and CC In comparison with the GG genotype the relative risk (OR [95% CI]) for diabetes was 1.06 [0.86-1.31]; p = 0.275 for the genotypes (GC + CC) . Our results show significant variations for the anthropometric and lipid parameters in the type 2 diabetes mellitus (T2DM) but were not associated with the polymorphisms at site of 4545.

VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Polymorphism of beta (â) casein gene and their association with milk production traits in Sahiwal and HF crossbred cattle

Present research work was carried out on 50 Sahiwal and 50 HF Crossbred cattle in the Department of Animal Genetics and Breeding of College of Veterinary Science and A H Jabalpur. During the research work the PCR amplified products of 121bp was digested by restriction endonuclease enzyme DdeI, which recognizes G^AATTC sites. The patterns evolved in the present study showed that the presence of one restriction site on one alleles and absence of restriction site on other alleles resulted in the appearance of three bands of 121, 86 and 35bp.This genotype was referred to as A1A2. When both strand had no restriction sites for the enzyme, only one band of 121bp was observed on the gel such genotype was designated as A2A2 type. The â- casein gene showed A2A2 and A1A2 genotypes were observed in Sahiwal and HF crossbred cattle. The genotypic frequencies of â-casein (CSN2)/ DdeI gene for A1A1, A1A2 and A2A2 are 0.00, 0.30, and 0.70 in Sahiwal and 0.00, 0.64 and 0.36 in HF crossbred cattle, respectively and the gene frequency A1 and A2 is 0.15 and 0.85 in Sahiwal and 0.32 and 0.68 in HF crossbred cattle. High frequency of A2 allele was observed in both the breeds of cattle under the study.

áS2 casein gene polymorphism and association with milk production traits in Malvi and Nimari cattle of Madhya Pradesh

The aim of this study was to know the polymorphic variants and their association with milk production traits in Malvi and Nimari cattle of Madhya Pradesh at áS2 casein gene (CSN1S2) gene locus. The PCR amplified products of 1267 base pair (bp) length were digested by restriction endonuclease enzyme EcoRV, which recognizes GAT^ATC sites. The 1267bp product was cut into two fragments of sizes 1150bp and 117bp. All the tested samples yielded different results. Absence of restriction site at both the alleles that resulted in the appearance of single compact bands of size 1267bp was referred to as genotype AA. The samples exhibiting three fragments (1267bp/ 1150bp/ 117bp) were denoted as genotype AB. The RFLP analysis carried out in above both of the breeds of cattle revealed dissimilar genotypic patterns. AA and AB genotypes were observed in Malvi and Nimari . The genotypic and gene frequencies of AA, AB and BB genotypes of áS2-casein gene (CSN1S2)/EcoRV locus was found to be 0.44, 0.56 and 0.00 in Malvi; 0.68, 0.32 and 0.00 in Nimari and the respective gene frequency for A and B alleles were found to be 0.72 and 0.28 in Malvi; 0.84 and 0.16 in Nimari. The frequency of A allele was found to be highest as compared to B allele in all the four breeds of cattle under the study. Chi-square values for testing correspondence between observed and expected genotypic frequencies at this locus were found to be non-significant in Malvi and Nimari breeds of cattle. The above result indicated that the populations of animals of above both breeds were in Hardy-Weinberg equilibrium at this locus. The association of polymorphic variants of áS2-casein gene (CSN1S2)/EcoRV in Malvi and Nimari breeds of cattle with milk yield per lactation (L), daily milk yield (L) showed the non significant difference was observed between mean MY(L) of AA and AB genotype of Malvi and Nimari and With respect of daily milk yield the Nimari. However, the Nimari breed showed significantly higher daily milk yield than the Malvi breed of cattle.

Whole-Genome-Sequence-Based Haplotypes Reveal Single Origin of the Sickle Allele during the Holocene Wet Phase

Five classical designations of sickle haplotypes are made on the basis of the presence or absence of restriction sites and are named after the ethno-linguistic groups or geographic regions from which the individuals with sickle cell anemia originated. Each haplotype is thought to represent an independent occurrence of the sickle mutation rs334 (c.20A>T [p.Glu7Val] in HBB). We investigated the origins of the sickle mutation by using whole-genome-sequence data. We identified 156 carriers from the 1000 Genomes Project, the African Genome Variation Project, and Qatar. We classified haplotypes by using 27 polymorphisms in linkage disequilibrium with rs334. Network analysis revealed a common haplotype that differed from the ancestral haplotype only by the derived sickle mutation at rs334 and correlated collectively with the Central African Republic (CAR), Cameroon, and Arabian/Indian haplotypes. Other haplotypes were derived from this haplotype and fell into two clusters, one composed of Senegal haplotypes and the other composed of Benin and Senegal haplotypes. The near-exclusive presence of the original sickle haplotype in the CAR, Kenya, Uganda, and South Africa is consistent with this haplotype predating the Bantu expansions. Modeling of balancing selection indicated that the heterozygote advantage was 15.2%, an equilibrium frequency of 12.0% was reached after 87 generations, and the selective environment predated the mutation. The posterior distribution of the ancestral recombination graph yielded a sickle mutation age of 259 generations, corresponding to 7,300 years ago during the Holocene Wet Phase. These results clarify the origin of the sickle allele and improve and simplify the classification of sickle haplotypes.

Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus gouramy Lac.) yang Mati pada Rentang Waktu Berbeda Menggunakan PCR-RFLP Gen Major Histocompatibility Complex Kelas II B

Gurami (Osphronemus gouramy Lac.) is a popular fish species among Indonesian people. Several Gurami strains have been cultivated by fish farmer, one of which is Gurami Soang. This strain is belived to have a faster growth rate compared to other strains. However, like other strains, the fingerling of Soang strain have also a low survival and suceptible to disease, especially that caused by Aeromonas hydrophila infection. It has been proved that seeds from a single spawning event show varibale disease resistance. The difference in resistance among individuals is suggested related to the difference in their genetic component. One of the genes responsible for resistance is Major Histocompatibility Complex (MHC) class II B gene. Variability in resistance can be analyzed by using PCR - RFLP technique. PCR-RFLP is a technique that can produce a specific DNA fragments by PCR, followed by cutting the PCR product using restriction enzymes to describe the presence or absence of restriction sites in DNA fragments. This research aims to determine genetic marker to differiantiate between resitant and irresistant individual of Gurami Soang infected by A. hydrophila which die at a different time priod based on PCR-RFLP MHC class IIB gene. The study used survey method with purposive random sampling. The Data of PCR-RFLP band patterns were analyzed descriptively. The result indicated that cutting of the MHC class II B gene using HinfI produce two RFLP bands with 300 bp and 100 bp length in all samples. Meanwhile, the MHC IIB gene was not cuted by PstI, HindIII, BamHI and EcoRI enzymes forall samples. These mean that MHC II gene in all individuals were monomorphic. Therefore,it can be concluded that there is no specific genetic marker to differentiate gurami soang individulas which was dying in different time periods.

Analysis of the association Hind III Polymorphism of Lipoprotein Lipase gene on the risk of coronary artery disease

Background and Aim: Coronary artery disease (CAD) is one of the leadin g causes of death and disability around the world. In teraction between genetic and environmental factors determines susceptibility of an individual to develop coronary artery disease. Lipoprotein lipase (LPL) play an important role in the metabolism of HDL-C (High Density Lipoprotein Cholesterol), LDL-C (Low Density Lipoprotein Cholesterol) and triglycerides (TG). Dysfunction of LPL as a result of genetic variants of lipoprotein lipase ge ne is associated with increased risk of CAD. The aim of the present study was to in vestigate the relationship between the risk of coronary artery disease and LDL -C, HDL-C and TG (triglycerides) levels by lipoprotein lipase gene Hind III polymorphism. Methods: A total of 202 subjects including 114 patients with coronary artery disease and 88 control participated in this study. The Hind III polymorphism of the lipoprotein lipase gene was determined by PCR- RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). In the presence and absence of restriction site, the genotypes are desc ribed H+/+, H-/- respectively. Results: In this survey, a significant association between the frequent H+/+ genotype and unfavorable TG levels was observed in our population. For the Hind III genotypes, within the healthy subjects (n=88), the H+/+ genotype was found in 67 individuals (58.8%), H-/+ genotype in 38 individuals (33.3%), and 9 individuals (7.8%) carried the H-/- genotype. Within the CAD group (n=114), 47 individuals (53.4%) with H+/+ genotype, 36 (41%) with H-/+ genotype, and 5 (5.6%) carried the H-/- genotype. Conclusion: There was no significant difference between the di stribution of LPL- Hind III genotypes and the healthy subjects and the patient s with CAD (P<0.05, 0. 645). The study of LPL genotypes confirms the exist ence of interrelations between TG levels (P<0.05), but this polymorphisms were not detected as independent risk factors for CAD (P<0.05).

ASSOCIATION OF ESTROGEN RECEPTOR Α PVU II AND XBAI GENE POLYMORPHISMS WITH BREAST CANCER RISK; RELATION TO THE AGE OF MENARCHE AND MENOPAUSE

The association of estrogen receptor-α (ER-α) genetic polymorphisms with the risk ofbreast cancer attracts much attention because ER functions as a hormone-dependenttranscriptional regulator, which in turn, plays a significant role in the development ofbreast cancer. This study was conducted to find if there is an association betweengenetic polymorphisms in the ER-α gene and breast cancer in Egyptian females andits relation to the age of menarche and menopause. A total of 50 breast cancerEgyptian women and 25 age-matched healthy controls were involved in the study.PvuII and XbaI polymorphisms of ER-α gene were genotyped by polymerase chainreaction restriction fragment length polymorphism. Pp/pp genotypes were found in84% of patients and in 56% of controls; and the homozygous wild PP genotype wasfound in 16% of patients, and 44% of controls. There was highly significant increasein the risk of breast cancer with the presence of PvuII restriction site (Pp/ppgenotypes) compared with absence of restriction site (PP genotype) (P = 0.008).There was statistically significant decrease in the age of menarche (P = 0.021) andinsignificant differences in the age of menopause of all participant women withpresence of PvuII restriction site. Xx/xx genotypes were found in 84% of patients andin 76% of controls; and XX genotype was found in 16% of patients and in 24% ofcontrols. There was insignificant difference in genotype frequency of the ER-α XbaIpolymorphism between patients and controls (P = 0.157). There was statistically veryhighly significant decrease in the age of menarche (P<0.001) and insignificantdifferences in the age of menopause of all participant women with presence of XbaIrestriction site. From the present study, it could be concluded that geneticpolymorphisms in the ER-α gene may play a role in the etiology of breast cancer inEgyptian women and may be a genetic determinants of the age of menarche.

DNA Damage-related Gene Expression as Biomarkers to Assess Low Dose Radiation Exposure

According to the UNSCEAR, the natural rays from the Sun and the Earth transmit about 2,4 mSv to each individual every year. Human activities expose us to an additional radiation dose (1,2 mSv/year), especially the techniques used in non-invasive medical imaging (radiography, CT scanners). Ionizing radiation can induce a large spectrum of DNA lesions, but under optimal DNA repair conditions, the principal residual lesions of importance are misrepaired double-strand breaks / 1 /. Predictive markers of intrinsic radio sensitivity in healthy individuals are needed in monitoring their occupational or environmental radiation exposure and may predict a patient's response to radiotherapy. Radiation protection requires a thorough understanding of low dose ionizing radiation. Currently extrapolation from high doses is necessary to estimate the effects of low doses. Furthermore, it is critically important to have an appreciation of the variation in individual responses to radiation among the human population / 2 /. Appearance of mutations consist one of more prominent consequence of the radiation action. The aim of our study consisted in the restriction fragment's length polymorphism (RFLP) analysis of pERT87-8/Tag1 and 16intron/Tag1 loci with determining of presence or absence of restriction site in the group of radiologists and in control group. It was demonstrated that the pERT87-8/Tag1 allele frequency on «mutant» chromosome was 2,4 fold higher than the frequency of this allele on «normal» chromosome (45,1% in compare with 18,5%, X2=27,7, df=1, p<0.01). Therefore, in radiologists was revealed statistically significant difference of the frequency of polymorphic site pERT87-8/Tag1 compared to healthy donors. As for 16intron/Tag1 system it have been elucidated that the without restriction allele frequency site on «normal» chromosome was 2,0 fold higher than the frequency of this allele on «mutant» chromosome (71,5% in compare with 34,8%) χ2=78,3, df=1, p<0,01). In the conclusion it is necessary to mention that there are significant difference in the frequency of the polymorph sites pERT87-8/Tag1 and 16intron/Tag1 in the group of radiologists in compare with the control group. Reference 1. Karine Bishay et al. DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation // Carcinogenesis, Vol. 22, No. 8, 1179-1183, 2001. 2. Robert C. Millikan et al. Polymorphisms in DNA Repair Genes, Medical Exposure to Ionizing Radiation, and Breast Cancer Risk // Cancer Epidemiology Biomarkers & Prevention Vol. 14, 2326-2334, October 2005.

Association between vitamin D receptor Fokl polymorphism and serum parathyroid hormone level in patients with chronic renal failure

We investigated the relationship between vitamin D receptor (VDR) start codon polymorphism and serum levels of PTH, calcidiol, and calcium in 64 Spanish patients with chronic renal failure (CRF). An exon 2 fragment of the VDR gene was amplified by PCR, and cleaved with the restriction enzyme FokI. The alleles were identified according to the digestion pattern obtained as F (absence of restriction site) and f (presence of restriction site). Genotype frequencies in the patient population were 54.7% FF, 28.1% Ff and 17.2% ff, vs 46.7% FF, 43.3% Ff and 10% ff in a healthy control population. The difference between the two populations was statistically significant (p<0.01). Within the patient population, mean serum PTH level in the FF group was significantly higher (159.77+/-25.69 pg/ml) than in both the Ff and ff groups (106.67+/-19.07 and 77.55+/-15.85 pg/ml, respectively; p<0.05). However there were no significant differences in serum levels of calcidiol or calcium among genotypes. These results suggest that FokI polymorphisms of the VDR gene may determine parathyroid response in CRF patients.

Mitochondrial DNA in the hybrid zone between Mus musculus musculus and Mus musculus domesticus: a comparison of two transects

We studied mtDNA introgression across the contact zone between Mus musculus musculus and M. m. domesticus in two independent transects in the Czech Republic and Bavaria, Germany. A total of 1270 mice from 98 localities in the Czech transect and 456 mice from 41 localities in the Bavarian transect were examined for presence or absence of a BamHI restriction site in the mt-Ndl gene. Using this simple mtDNA marker, variants that belonged to the M. m. domesticus lineage (presence of restriction site) could be unequivocally distinguished from those belonging to the M. m. musculus lineage (absence of restriction site). The extent of introgression of mtDNA, three autosomal allozymes and the X chromosome was compared. The introgression of X markers was more limited than was that of the allozymes and mtDNA. In the Czech transect, the centre for the mtDNA dine was shifted about 3.6 km to the west relative to the X chromosome dine, with asymmetric introgression from M. m. musculus to M. m. domesticus. Interestingly, in the Bavarian transect, the centre of the mtDNA dine was shifted about 10.9 km to the east relative to the X chromosome dine, with asymmetric introgression from M. m. domesticus to M. m. musculus, opposite in direction to that observed in the Czech transect.

Restriction mapping of the IGS region in Alternaria spp. reveals variable and conserved domains

Accurate identification of Alternaria spp. is dependent upon the production of diagnostic morphological characters under defined cultural conditions and the proper assessment of character variation. This process is often compromised by variation in laboratory facilities and technical expertise. To assist taxon identification and phylogenetic studies, restriction site information from the intergenic spacer (IGS) region of nuclear rDNA was evaluated. Restriction maps were constructed from 15 species of Alternaria and Stemphylium botryosum (telemorph Pleospora herbarum) for 11 restriction enzymes using a new method for restriction mapping based on differential priming of IGS amplicons. IGS fragment size varied among species from 2.2-3.9 kb. Based upon restriction site homology among closely-related and more distantly related species, the IGS region could be divided into conserved and variable domains. The conserved domain was approximately 0.75 kb in size and was located at the 3' end of the IGS region. Restriction site homology within this region was very high, especially among closely related taxa. The remainder of the region comprised the variable domain, which encompassed considerable differences in size and restriction sites among taxa. The presence or absence of restriction sites among taxa was analyzed using methods of neighbor-joining. Phylogenetic relationships based on this method were concordant with those previously resolved based upon other methods and other genomic regions.

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.

DNA technology for the detection of common genetic variants that predispose to thrombophilia.

With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness.

Molecular genetic evidence for the post‐Pleistocene divergence of populations of the arctic‐alpine ground beetle Amara alpina (Paykull) (Coleoptera: Carabidae)

Summary Aim This study examines the hypothesis that the biogeographic history of a species is reflected in the distribution of molecular genetic diversity and the phylogenies of extant populations. Location Populations of arctic‐alpine ground beetle Amara alpina were analysed from Beringia (Alaska and northernmost British Columbia), the Hudson Bay region, the northern Appalachian Mountains, and the central Rocky Mountains of North America. Methods Mitochondrial restriction site variation of specimens from twenty‐two populations were assayed by using radioactively labelled mtDNA to probe Southern membranes containing restriction enzyme digested total DNA. Restriction sites were mapped and genetic distances were calculated by pairwise comparison of presence and absence of restriction sites. Genetic distances were used in a molecular analysis of variance and to construct a minimal spanning tree. Parsimony methods were used to investigate the phylogenetic relationships between the haplotypes. These results were compared to an existing model for postglacial dispersal based on fossil and modern occurrences of arctic‐alpine beetles. Results Among the twenty‐two populations, fifteen haplotypes were detected. Genetic variation within each of the four regions corresponded to that expected from the palaeontologically based model. Beringian populations were the most genetically diverse. In contrast, no restriction site variation was observed in populations from the Hudson Bay region. Intermediate amounts of variation were observed in alpine populations of the Rocky and Appalachian Mountains. Maximum parsimony and cluster analysis provide evidence that at least two ancestral haplotypes existed in the Southern refugium from which the Rocky and the Appalachian Mountains populations were founded. Main conclusions The genetic results are generally consistent with the palaeontologically based model. The diversity of Beringian populations is consistent with this region having been continuously inhabited by Amara alpina throughout the Pleistocene. The Hudson Bay region was not deglaciated until about 6000 years, and its populations have no restriction site variation. The molecular genetic data support the interpretation that the Hudson Bay region was colonized from Beringia based on the occurrence of the same haplotype in both regions.

Association of vitamin D receptor gene polymorphism with multiple sclerosis in Japanese.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D, exerts an immunosuppressive effect and can completely prevent experimental autoimmune encephalomyelitis (EAE). 1,25(OH)2D3 exerts most of its actions only after it has bound to its specific nuclear receptors. To investigate the possible role of vitamin D receptor gene (VDRG) polymorphism in susceptibility to or disease-modulation of MS, we evaluated 77 Japanese patients with 'conventional' MS and 95 controls. A VDRG allelic polymorphism was assessed by Bsm1 endonuclease restriction after specific PCR amplification. Genotypic polymorphism was clearly defined as BB (absence of restriction site on both alleles), bb (presence of restriction site on both alleles), or Bb (heterozygous). We found overexpression of the b allele (92.9 vs. 84.2%: P=0.0138) and homozygote bb (85.7 vs. 71.6%; P=0.0263) in MS patients compared with controls. The results indicate for the first time an association of MS with VDRG polymorphism, which may be involved in pathogenesis of MS, or in the linkage disequilibrium of VDRG to another pathogenic gene loci. The role of VDR gene polymorphism should be further studied in other populations, and the distribution of other polymorphism, such as Apa I, Taq I, should be also analyzed to confirm another susceptibility gene for MS and to obtain more adequate strategies for treatment of MS.

Vitamin D receptor gene polymorphisms affect secondary hyperparathyroidism in hemodialyzed patients

Adynamic bone disease unrelated to aluminum deposition, with low parathyroid hormone (PTH) levels, has increased in patients with end- stage renal failure. Some patients present with severe secondary hyperparathyroidism despite calcitriol administration and phosphate restriction. Because therapeutic and environmental factors are now similar among hemodialyzed patients, the variable incidence of secondary hyperparathyroidism may be caused by genetic heterogeneity. To examine this possibility, we analyzed restriction fragment length polymorphisms of the vitamin D receptor (VDR) gene in 877 Japanese hemodialysis patients. VDR allelic polymorphism was determined by the method of Morrison et al. Polymerase chain reaction (PCR) amplification and a BsmI endonuclease restriction site at the 5' end of the VDR gene defined BB (absence of restriction site on both alleles), Bb (heterozygous), or bb (restriction site on both alleles). The mean serum PTH level was lower in BB patients (86 +/- 102 pg/mL) than in bb patients (148 +/- 217 pg/mL; P < 0.05). The serum osteocalcin level was also lower in BB than in bb patients (P < 0.05). If results were re- analyzed excluding patients with a history of dialysis exceeding 10 years or those with non-insulin-dependent diabetes mellitus (NIDDM) or who had undergone parathyroidectomy, the differences in serum PTH levels were greater. However, there was no significant difference in serum PTH levels among the VDR genotypes, only for patients with NIDDM. The present study shows that patients with the b allele for the VDR gene have more severe secondary hyperparathyroidism than patients without the b allele. However, NIDDM or a long history of hemodialysis has a stronger power to influence PTH secretion. (Am J Kidney Dis 1998 Sep;32(3):464-9)

Relationships amongOmphalotus species (Paxillaceae) based on restriction sites in the ribosomal ITS1-5.8S-ITS2 region

Genetic relationships for several species of the fungusOmphalotus were estimated by comparing the presence or absence of restriction sites in the ITS1-5.8S-ITS2 region of nuclear ribosomal DNA. Results placeO. olearius, O. subilludens andO. olivascens in a single clade.Omphalotus illudens, usually thought to be related toO. subilludens, was placed in a second clade, more closely related toO. nidiformis. Omphalotus mexicana is distinct from all other examinedOmphalotus species.

PCR screening for carriers of bovine leukocyte adhesion deficiency (BLAD) and uridine monophosphate synthase (DUMPS) in Argentine Holstein cattle.

BLAD (Bovine Leukocyte Adhesion Deficiency) and DUMPS (Deficiency of Uridine Monophosphate Synthase) are monogenic autosomal, recessive inherited diseases of Holstein cattle. Single nucleotide changes (point mutations) responsible for the genetic disorders were detected by polymerase chain reaction coupled with restriction fragment length polymorphism assays (PCR-RFLP). Using oligonucleotide primers, DNA fragments of predicted sizes were amplified, and the products' specificity was assessed by nucleotide sequencing. Mutations were detected in DNA samples from bovine blood and semen by the presence or absence of restriction sites within the PCR amplification products (Taq I, Hae III for BLAD, Ava I for DUMPS). The test included 104 bulls and 950 cows of Argentinean Holstein breed. Defective alleles frequencies were as follows: 2.88% BLAD in bulls used in artificial insemination, 1.79% in cows; 0.96% DUMPS in bulls and 0.11% in cows.

Sensitivity of naturally occurring coliphages to type I and type II restriction and modification.

Protection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution. There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems. Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria. To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems. The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage. The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate. On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E. coli and its accessory elements.