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Hemophilia A is an X-linked recessive bleeding disorder caused by a quantitative or qualitative deficiency of blood coagulation factor VIII (FVIII). ARMS (amplification refractory mutation system) primers were designed to determine allele frequencies of three FVIII gene linked markers, IVS7 nt 27 G/A SNP, BclI/intron 18, and HindIII/intron 19 among 85 normal Iranian women from unrelated families. Then same method was applied to perform carrier detection for hemophilia A families. The allele frequencies of IVS7 nt 27 "G"/"A" allele, BclI "T"/"A" allele, and HindIII "C"/"T" allele among normal women were 0.88/0.12, 0.52/0.48, and 0.48/0.52, respectively. The three polymorphisms were found to be in strong linkage disequilibrium, which decreased the overall heterozygosity to 51%. Twenty-one women from 15 unrelated hemophilia A families were referred to us for hemophilia A carrier detection. Taking advantage of these three biallelic polymorphisms in conjunction with multiallelic St14 VNTR (locus DXS52), IVS13 (CA)n STR, and IVS22 (CA)n STR, carrier status was determined in 16 women (16/21 or 76% of the at-risk women) from 11 families (11/15 or 73% of the families). The used ARMS methods are rapid and can easily be applied in conjunction with other FVIII gene linked polymorphisms for indirect mutation detection of hemophilia A where they are informative.

The over-expression of amyloid precursor protein (APP) gene in certain areas of the brain indicates abnormalities in gene regulation as an important factor for the development of Alzheimer's disease (AD). We have reported recently that APP mRNA expression is lower in female as compared to male and is regulated by sex steroids. As methylation of promoter is crucial for such regulation, we have used isoschizomeric restriction enzymes MspI and HpaII to analyze the pattern of APP promoter methylation in the cerebral cortex of intact, gonadectomized, testosterone- and estradiol-treated adult and old mice of both sexes. Southern blots of DNA digested with HindIII/MspI or HindIII/HpaII from different groups of mice were probed with a 1.27-kb DIG-11-dUTP labeled APP promoter fragment. The results revealed four distinct bands of 315 bp, 596 bp, 911 bp, and 2.6 kb by MspI, a single band of 2.6 kb by HpaII in all groups and an additional 2.9 kb in intact and estradiol treated old male and testosterone-treated adult female mice. The intensity of 2.6-kb band was relatively lower in intact female as compared to male and varied with different treatments in both ages, indicating the modulation of methylation. Thus, these findings showed that APP promoter methylation is higher in female and differentially regulated by sex steroids in the mice cerebral cortex, suggesting a strong correlation between promoter methylation and transcriptional silencing of APP.

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases Xbal, Kpn I and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism. (C) 2005 Elsevier B.V. All rights reserved.

Fasting plasma glucose (FPG) levels are usually tightly regulated within a narrow physiologic range. Variation of FPG levels is clinically important and is strongly heritable. Several lines of evidence suggest the importance of the oestrogen receptor α (ER-α) and osteocalcin (also known as BGP, for bone Gla protein) in determining FPG; however, whether their polymorphisms are associated with FPG variation is not well understood.To investigate whether ER-a PvuII and BGP HindIII genetic polymorphisms and their potential interaction are associated with FPG variation.The study subjects were 328 unrelated pre-menopausal Chinese women aged 21 years and over (mean age ± SD, 33.2 ± 5.9 years), with an average FPG of 4.92 (SD = 0.81). All subjects were genotyped at the ER-α PvuII and BGP HindIII loci using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP).The ER-α PvuII genotypes were significantly associated with FPG (p = 0.007). In addition, a significant interaction was observed of the ER-α PvuII polymorphism with BGP HindIII polymorphism on FPG variation (p = 0.013), although the BGP HindIII polymorphism was not shown to be individually associated with FPG.The PvuII polymorphism of the ER-α gene and its potential interaction with the HindIII polymorphism of the BGP gene were associated with FPG in pre-menopausal Chinese women.

The aim of this research was to identify and localise re-peat sequences in Korean cattle (Hanwoo), which may be a basis for future genetic studies. To achieve this, a Ha n-woo BAC library containing a total of 150,000 clones with an average size of 130–140 kb was constructed us-ing two different restriction enzymes Eco RI and Hind III. A total of 2,794 clones were sequenced successfully at both 5′ and 3′ terminals using RP2 and T7 primers, ge n-erating 5,588 mate-pairs end sequences amounting to a total of 3.3 × 10

Problem Erythrocyte complement receptor type 1 (E-CR1) is the main immune complex clearance mechanism in humans. Decreased E-CR1 expression is noted in certain inflammatory disorders. Recent evidence implicates inflammation in the pathogenesis of preeclampsia. We investigated whether E-CR1 is decreased in preeclampsia. Method of study E-CR1 protein expression was quantified by radioimmunoassay. Plasma concentration of soluble CR1 was quantified using a specific enzyme linked immunosorbent assay. Quantitative genotypes were evaluated by HindIII restriction fragment length polymorphism analysis. Results E-CR1 expression was reduced in patients with preeclampsia. Lack of neoantigen expression (indicative of enzymatic cleavage of CR1) or elevated plasma-soluble CR1 was evidence against an acquired loss of E-CR1. Genotype analysis revealed a higher frequency of a CR1 allele associated with low E-CR1 expression in preeclampsia when compared with normal pregnant controls. Conclusions E-CR1 expression is decreased in preeclamptic patients and levels correlate with severity of disease. This condition may have a genetic basis in some patients.

BACKGROUND: Women with recurrent pregnancy loss (RPL) and T-helper (Th)1-type immunity to trophoblast antigens have an increased frequency of the IL1B-511*1 promoter variant. Since CD46 gene products also regulate maternal immune responses including Th1 immunity, we investigated whether CD46 gene polymorphisms are also associated with RPL in women with and without Th1 immunity to trophoblast, and the possibility of a synergistic effect with the IL1B-511*1 promoter variant. METHODS: A case-controlled study was performed to document HindIII site polymorphism in intron 1 of the CD46 gene in 131 women with RPL and 72 fertile controls. Clinical information, Th1-type immune responsiveness to trophoblast in women with RPL history, and IL1B promoter allelo-types for this cohort were documented in a previous study. RESULTS: The frequency of the CD46H*2 allele and CD46H*2 homozygosity were significantly increased in women with RPL compared with fertile controls (P

A total of nine Trichinella nativa isolates were compared by amplified fragment length polymorphism (AFLP). Four hundred nanograms of genomic DNA from a pool of 10--20 larvae were digested using HindIII and MseI restriction endonucleases. Of the 16 primer combinations initially tested, Hind-C and Mse-C primers showed rich polymorphism with approximately 40--90 bands in the range of 30--270 bp. Genetic similarities were estimated visually. AFLP provided discriminatory banding patterns and may therefore be used as a method for detecting variation in T. nativa populations. However, the heterogeneous patterns obtained from pooled samples emphasize the need for further development of the sampling and numerical analysis of the patterns for epidemiological and taxonomical interpretation.

Objectives: Osteoporosis constitutes a serious health problem in old people. Bone mineral density (BMD) is determined by multiple genetic and environmental factors. The genetic control of BMD and osteoporosis is better understood in women, but much less in men. The present study evaluated the relationship of COL1A2, BGP, IL-6, AHSG and PTH genes defined by Msp I, Hind III, BsrB I, Sac I and BstB I restriction enzymes, respectively, with BMD in Chinese males. Methods: A total of 258 unrelated healthy Chinese men aged 50–80 years were recruited. BMD at spine (L1–4) and femoral neck were measured by a Hologic 2000+ densitometer and adjusted by significant covariates of age, height and weight. All the subjects were genotyped at the upper five polymorphic sites by PCR-RFLP procedure. Results: We revealed significant association of the AHSG gene with the spine BMD ( P = 0.006), even after adjusting for multiple testing in our study. Carriers of 1 * 1 and 2 * 2 genotypes of AHSG gene had, respectively, ∼5.1 and 8.1% higher spine BMD than those of 1 * 2 genotype. For the other four genes, no evidence of association was found ( P > 0.10). No significant evidence of gene-by-gene interaction was found by two-way factorial ANOVA on the BMD variation. Conclusions: The results suggest that the AHSG gene is associated with the spine BMD in Chinese men. The present study represents the first effort to simultaneously investigate the effects of single gene locus as well as gene-by-gene interactions of multiple genes on BMD variation in Chinese men.

An improved DNA-mediated transformation system for nematode-trapping fungus Arthrobotrys oligospora based on hygromycin B resistance was developed. The transformation frequency varied between 34 and 175 transformants per microg linearized DNA and 93% of the transformants were stable for drug resistance when tested 100 randomly selected transformants. More than 2000 transformants were obtained by transformation of the fungus with pBChygro in the presence of HindIII and among them, one, YMF1.00110, which lost its ability of forming predacious structure, was isolated. Southern analysis showed that the plasmid DNA had integrated into the genome of all tested transformants (including YMF 1.00110) except one. The transformant tagged with hph gene could be re-isolated and quantified from dung samples based on the resistance of hygromycin B. All the results suggested that the method of restriction enzyme mediated integration (REMI) should facilitate not only the insertional mutagenesis for tagging and analysis genes of interest but also the ecological investigation of tagged fungi in a given environment.

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analysed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei species. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of Lactobacillus casei and Lactobacillus paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (=ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separated subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (=ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.

The polymorphism of nuclear ribosomal DNA has been studied in male German cockroaches. The RFLP analysis has been used to identify seven types of HindIII fragments, the variation of which may serve as the basis for the differentiation of laboratory strains with respect to some population indices, including the average number of fragments per animal (N), the proportion of polymorphic loci (P), and the average pairwise similarity (APS). The interpopulation variation (FST) calculated for nine haplotypes is 53.85%.

Objective : To investigate the association of lipoprotein lipase gene intron 8 polymorphisms and Essential Hypertension in Han race Chinese. Methods and results : 116 patients with Essential Hypertension were enrolled and another 116 normal people were served as controls. All cases were examined for the genotypes of intron 8 in lipoprotein lipase gene by the methods of polymerase chain reaction restriction fragment length polymorphism, and the serum lipoprotein levels were also observed. Results showed that body mass index blood pressure and the serum triglyceride level were obviously increased in the Essential Hypertension group. The genotype and allele frequency of intron 8 in lipoprotein lipase in the Essential Hypertension group showed obvious differences compared with the control group. Serum triglyceride levels were higher in the patients with H + H + genotype than in those in non H + H + genotype of intron 8 in lipoprotein lipase by Hind III digestion. The systolic blood pressure showed a decreasing tendency among the H + H + genotype, the H + H − genotype and the H − H − genotype individuals. Conclusion : The results suggest that lipoprotein lipase may be an important genetic factor associated with the Chinese Han race Essential Hypertension patients. The polymorphisms of intron 8 in lipoprotein lipase influence the blood-lipid metabolism, induce blood vessel rebuilding and play an important role in the invasion and development of Essential Hypertension.

Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the cause of considerable economic losses in the poultry industry. Molecular differentiation of avian Mycoplasma strains may be helpful in tracing infections and in the evaluation of implemented intervention strategies. Amplified Fragment Length Polymorphism (AFLP) has shown to be a powerful typing technique but the application for poultry Mycoplasma strains is very limited. The aim of this study was to evaluate the reproducibility and discriminatory power of AFLP HindIII/HhaI and AFLP BglII/Mfel for the inter- and intraspecies differentiation of avian mycoplasmas and to compare these test characteristics with digitalized Random Amplified Polymorphic DNA (RAPD) analysis. The reproducibility of RAPD, AFLP HindIII/HhaI and AFLP BglII/Mfel was 50-100, 97-98 and 86-99%, respectively. RAPD and both AFLP enzyme combinations were able to differentiate between five avian Mycoplasma species. For AFLP, five MG and four MS clusters could be identified. The phylogenetic tree for both enzyme combinations was comparable. For RAPD, four MG clusters could be identified. For MS, however, due to the poor reproducibility of the RAPD technique, no clear genogroups could be identified. On basis of the results of this study it can be concluded that AFLP is a powerful technique for the genotyping of avian mycoplasmas and that, although AFLP HindIII/HhaI generated patterns with less fragments, the final results showed homologous results.

Large-insert bacterial artificial chromosome (BAC) libraries, plant-transformation-competent binary BAC (BIBAC) libraries, and simple sequence repeat (SSR) markers are essential for many aspects of genomics research. We constructed a BAC library and a BIBAC library from the nuclear DNA of chickpea, Cicer arietinum L., cv. Hadas, partially digested with HindIII and BamHI, respectively. The BAC library has 14,976 clones, with an average insert size of 121 kb, and the BIBAC library consists of 23,040 clones, with an average insert size of 145 kb. The combined libraries collectively cover ca. 7.0 x genomes of chickpea. We screened the BAC library with eight synthetic SSR oligos, (GA)10, (GAA)7, (AT)10, (TAA)7, (TGA)7, (CA)10, (CAA)7, and (CCA)7. Positive BACs were selected, subcloned, and sequenced for SSR marker development. Two hundred and thirty-three new chickpea SSR markers were developed and characterized by PCR, using chickpea DNA as template. These results have demonstrated that BACs are an excellent source for SSR marker development in chickpea. We also estimated the distribution of the SSR loci in the chickpea genome. The SSR motifs (TAA)n and (GA)n were much more abundant than the others, and the distribution of the SSR loci appeared non-random. The BAC and BIBAC libraries and new SSR markers will provide valuable resources for chickpea genomics research and breeding (the libraries and their filters are available to the public at http://hbz.tamu.edu).

Results of studying plasmid pAH36 in strain Aeronomas hydrophila IBRB-36 4CPA are presented. Plasmid pAH36 possesses BamHI, PstI, and HindIII restriction sites and is 5.4 kb in size. The plasmid was shown to contain genes for catabolism of chlor-substituted phenoxyacetic acids.

Two closely related lactic acid bacteria, Lactobacillus sakei and Lactobacillus curvatus, are very difficult to be rapidly differentiated. Here we report multiplex polymerase chain reaction (PCR)-based restriction enzyme analysis that is useful for rapid and reliable identification of these two species. This method employs both polymerase chain reaction (PCR) and restriction enzyme analysis (REA). First, multiplex-PCR using three primers that were designed from 16S rDNA sequence produces two bands, a 433-bp and a 623-bp band. A 433-bp band represents only L. sakei and L. curvatus among lactobacilli and genetically related bacteria, and a 623-bp band is used for further identification by restriction analysis. Second, restriction analysis of 623-bp band using Hind III restriction enzyme discriminates L. sakei from L. curvatus. This method could identify 28 strains as L. sakei or L. curvatus, which were frequently isolated from kimchi, a traditional fermented cabbage product in South Korea. Therefore, these results suggest that this method is simple, rapid, and reliable for the identification of L. sakei and L. curvatus species.

The objective of this study was to assess the frequency of factor VIII (FVIII) gene intron 1 and intron 22 inversions and the informativeness of polymorphic markers for the genetic diagnosis of patients with haemophilia A (HA). Fifty unrelated patients with HA were first assessed for the intron 1 and intron 22 inversion mutations. Inversion-negative families were then screened for the bi-allelic intragenic markers--intron 7 G-->A polymorphism, HindIII site in intron 19 and XbaI site in intron 22 and the multiallelic dinucleotide CA repeat alleles in introns 13 and 22. The extragenic, multiallelic VNTR DXS52 (st14) was also analysed. Intron 22 inversion mutation was found in 38% (n = 19) of all patients and 46% of those with severe HA. Intron 1 inversion was found in one (2%) patient. Of the 30 inversion-negative families, XbaI site polymorphism was the single most informative marker (70%, n = 21/30) followed by HindIII (60%, n = 18/30), intron 13 CA repeats (56.66%, n = 17/30), intron 22 CA repeats (50%, n = 15/30), DXS52 VNTR (23.33%, n = 7/30) and intron 7 G-->A polymorphism (6.66%, n = 2/30). The combined use of these markers was informative in 92% (n = 46/50) of HA families. Based on the informativeness of these markers a comprehensive algorithm has been proposed for genetic diagnosis of HA in India.

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.

resultant fragments is further destroyed at its unique restriction-purification site. The crude digestion products are ligated in situ to generate a single plasmid containing the two fragments that can be selected for directly. Two of the two-fragment plasmids are then ligated to make a plasmid with a four-fragment insert, and the process is continued recursively to lengthen the size of the insert in each cycle. Although the method has been developed to stitch together synthetic genes, a modified version could as well be used to ligate multiple DNA fragments from other sources (e.g., PCR). All enzymes were obtained from New England Biolabs (Beverly, MA, USA) and used as recommended. Molecular biological techniques were performed using standard protocols (2). Synthetic DNA fragments were designed and prepared by a modification of reported methods (3–5). A linker duplex for uracil-DNA glycosylase-based ligation-independent cloning (UDG-LIC) (6,7) comprised of oligonucleotides UDG-L1 and UDG-L2, which contained a 5′ overhang [nucleotides (nt) 1–4] compatible with HindIII and a 3′ EcoRI overhang (nt 66–69), was introduced into HindIII/EcoRI-digested pUC18 to give pKOS293-118-72. The linker contained a SacI site (nt 36–41), a NotI site (nt 6–13), and two N. BbvCIA sites (nt 14–20 and 58–64), and upon introduction destroyed the HindIII site. The chloramphenicol resistance gene (CmR) was amplified from pACYC184 with the primers Cm-forward and CmDNA ligation by selection

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.

A strain of Cucumber mosaic virus (CMV) was isolated from a weed, water chickweed (Stellaria aquatica), growing in the pepper field in Chunchon, Korea. This isolate, CMV-Sa, was differentiated from other CMVs based on biological properties and nucleotide sequence analysis of the coat protein (CP) gene. CMV-Sa showed different reactions to all the tested plants, except Capsicum annuum and Cucumis sativus, when compar-ed with those of CMV-Mf (subgroup I) and CMV-PaFM (subgroup II). Remarkably, in Nicotiana tabacum cvs. Samsun, Xanthi-nc and Ky-57, CMV-Sa induced local necrotic ring spots on the inoculated leaves and venal wave pattern and mosaic on the upper leaves. RNA analysis, serology, and RT-PCR of CP gene showed that CMV-Sa belonged to subgroup I of CMV. However, restriction enzyme analysis of the cDNA using AluI, HhaI, HincII, HindIII, HinfI and MspI showed that CMV-Sa was distinct from that of CMV-Mf. Based on comparison of the nucleotide of CP gene and deduced amino acid sequences between other CMV strains, CMV-Sa was closely related to CMV-Mf with 93.7% and 97.2 % identity, respectively.

Background: Familial LPL deficiency is a rare inborn error of metabolism caused by mutational change within the LPL gene, which leads to massive hypertriglyceridemia. Methods: The underlying molecular defect in a boy of Croatian descent was studied by SSCP analysis, DNA sequencing and finally confirmed by RFLP. Results: DNA analysis showed the child to be a homozygote and his parents heterozygotes for TGG→CGG change in codon 86 of the LPL gene, which leads to W86R amino acid substitution. DNA sequence analysis also showed a silent mutation in the third exon of father's DNA, V108V. Determination of some LPL gene polymorphisms showed the child and his parents to have HindIII/H+H+ and both S447 wild-type alleles, whereas for PvuII the parents had P+P− and the child P+P+ genotype. Conclusions: In this case, W86R mutation was the reason for the production of nonfunctional enzyme and consequently triacylglycerol (TG) exceeding 15 mmol/l. This implies the risk of frequent episodes of acute pancreatitis. Decreased LPL activity leads to elevated triacylglycerol levels and reduced HDL-cholesterol, both risk factors for the development of coronary artery disease. LPL genotyping especially of young patients with hypertriglyceridemia is therefore necessary and justifiable.

A collection of 113 epidemiologically unrelated Streptococcus agalactiae strains were studied (group B streptococcus; GBS): they belonged to different serotypes and were isolated from pregnant women in China and Russia. The insertion sequence ISSa4 was found in 21 of 113 strains (18,6%). All of the strains with ISSa4 belonged to serotypes II and II/c and were characterized by the presence of IS 1381 and IS 861 as well as the absence of IS 1548 and GBSi1. All of the strains with ISSa4 possessed both bca and bac virulence genes coding for α and β antigens, respectively. Among 21 ISSa4-positive strains, 13 different Hin dIII patterns (D1 to D13) hybridizing with an ISSa4 probe were found. One of them (D13) contained a single Hin dIII hybridization fragment 6.5 kb in size that was found to be specific for all ISSa4-positive GBS strains. Multiple target sites for insertions of ISSa4 were identified and included a putative pathogenicity island, “housekeeping” genes, and intergenic regions, as well as the genes for hypothetical proteins. No significant similarity was observed in the sequences of the target genes for ISSa4 insertions, in the relative location of the target genes on the chromosome, or the biological functions of the encoded proteins. The possible significance of ISSa4-based differentiation of the strains and the presence of possible “hot spots” for insertions of ISSa4 in GBS genome are discussed.

In a recent study, a large proportion of multi-drug-resistant (MDR) Acinetobacter baumannii strains that were isolated from hospitalized patients in the Czech Republic was found to belong to two major groups (A and B). These groups appeared to be similar to epidemic clones I and II, respectively, which were identified previously among outbreak strains from north-western European hospitals. The aim of the present study was to assess in detail the genetic relatedness of Czech A. baumannii strains and those of epidemic clones I and II by using ribotyping with HindIII and HincII and by AFLP fingerprinting. The study collection included 70 MDR strains that were isolated in 30 Czech hospitals in 1991–2001, 15 susceptible Czech strains from 1991 to 1996 and 13 reference strains of clones I and II from 1982 to 1990. One major HindIII/HincIII ribotype (R1-1) was observed in 38 MDR Czech strains and eight reference strains of clone I, whereas another major ribotype (R2-2) was observed in 11 MDR Czech strains and in three reference strains of clone II. A selection of 59 Czech strains (representative of all ribotypes) and the 13 reference strains were investigated by AFLP fingerprinting. At a clustering level of 83 %, two large clusters could be distinguished: cluster 1 included all reference strains of clone I and 25 MDR Czech strains, whilst cluster 2 contained all reference strains of clone II and 11 MDR Czech strains. There was a clear correlation between the groupings by AFLP analysis and by ribotyping, as all strains with ribotype R1-1 and four strains with slightly different ribotypes were found in AFLP cluster 1, whereas all strains with ribotype R2-2 and seven strains with similar ribotypes were in AFLP cluster 2. Thus, 41 and 21 MDR Czech strains could be classified as belonging to clones I and II, respectively. The remaining eight MDR and 15 susceptible strains were highly heterogeneous and were distinct from clones I and II by both AFLP fingerprinting and ribotyping. These results indicate that the two predominant groups observed among MDR Czech A. baumannii strains from the 1990s are genetically congruent with the north-western European epidemic clones that were found in the 1980s. Recognition of these clinically relevant, widespread clones is important in infection prevention and control; they are also interesting subjects to study genetic mechanisms that give rise to their antibiotic resistance and epidemic behaviour.

Three new genes of Cryptosporidium parvum were cloned, including a gene encoding methionine aminopeptidase, one encoding chaperonin containing T-complex protein 1 delta (TCP-1 delta) and one with unknown function. DNA sequence analysis indicated that these genes are quite conserved, but there were some base pair differences between genotype I and genotype II isolates. These differences were confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 3 genes from 41 isolates collected from different hosts and geographical origins. In brief, the band patterns generated by endonuclease Hind III or Hinf I restrictions of the gene of methionine aminopeptidase, Sac I restriction of the gene of chaperonin, or Ava II restriction of the unknown gene could differentiate the isolates of C. parvum into genotype I and genotype II. PCR primers based on these genes amplified only C. parvum genes. Even a single oocyst was detectable with these PCR primers. Thus the results provided further evidence that genotype I and genotype II are distinct, and our three new primers can be used to detect and characterize C. parvum isolates with high sensitivity.

SUMMARY The Pervenets mutation raises the oleic acid content (OAC) from about 25% (LO linoleic sunflower) to over 75% (HO sunflower) in sunflower seed oil whatever the genetic backgrounds of the genotypes: fixed mutant lines, hybrid between fixed mutant lines and, surprisingly, both direction cross hybrids between high oleic × linoleic and linoleic × high oleic lines. Here we develop the methodology to point out the coincidence between the mutation and the oleate-desaturase locus. The mutation function is modeled to explain the phenotype. A series of sunflower genotypes developing embryos between 12 and 24 days after pollination without or carrying the Pervenets mutation was used to compare oleate-desaturase transcript accumulation. This revealed the presence and absence of the transcript, in normal LO and Pervenets HO embryos, respectively, whereas stearate-desaturase transcript accumulation was equivalent in both types. The RFLP approach using oleate-desaturase cDNA as a probe has revealed that in comparison with the linoleic sunflower, those carrying Pervenets mutation displayed, besides a common 5.8 kb EcoRI fragment, an extra 8 kb EcoRI fragment and, with HindIII, the shift from 8 kb to 16 kb of a HindIII fragment. The common 5.8 kb EcoRI fragment to Pervenets and normal sunflower carries an oleate-desaturase gene. The insertion unique to the sunflower carrying Pervenets mutation has to carry oleate-desaturase sequences. The genetic studies of this mutation have lead to contradictory results in the inheritance pattern of the HO trait due to variable mutation expression and several genetic factors affecting the HO level. Thus, it appeared important to study the inheritance in a set of recombinant inbred lines. Moreover, we pointed out that the presence of the mutation is not sufficient to induce the HO phenotype since a suppressor of the mutation “supole” may mask the HO trait. The supole allele is revealed in half of the HO RI lines, and consequently its accurate mapping is difficult. Thus conjunction of the molecular and genetic methods and of material construction was required to solve this problem and to provide efficient tools to breed HO sunflower.

The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.

The structure of heterochromatin in mitotic chromosomes in Prochilodus lineatus was investigated by di- gesting the chromosomes with three restriction enzymes, BamHI, DdeI and HindIII. The results suggested the exist- ence of distinct classes of highly repetitive DNA among the A-chromosomes. Also, a difference was revealed between the heterochromatin of the complement of A-chromosomes and that of the B-chromosome; the latter could only be cleaved by HindIII.

Randomly amplified polymorphic DNA (RAPD) and automated amplified-fragment length polymorphism (AFLP) techniques with fluorescently labelled primers were used to type non-serotypable Haemophilus influenzae (NTHI) isolates. Eighty-seven isolates from healthy children attending day-care centres or living at orphanages in southern Poland were investigated. Through comparison of the AFLP data with RAPD analysis, it has been concluded that the discriminatory power of AFLP for NTHI typing is higher than RAPD. Generally, the NTHI isolates analysed were highly heterogeneous, as detected with a HindIII/TaqI AFLP genotyping scheme on intra/inter similarity levels of 94 and 96 % using Pearson's correlation coefficient. The range of similarity values found for isolates from children permanently residing at a particular day-care centre was much wider than that for isolates from orphanages. AFLP can efficiently access NTHI strain diversity and can monitor their turn-over for comparative typing in local and inter-local epidemiological investigations.

Single-copy RFLP markers were developed for the root endophytic fungus Phialocephala fortinii. After an initial screening of 40 probes with four restriction enzymes, EcoRV and HindIII were chosen to analyse 31 strains of P. fortinii and nine strains of a closely related morphotype (Type 1) which have known ISSR-PCR or allozyme phenotypes. Type 1 isolates and a single genetically different isolate with Type 1 like morphology, a representative of a possible third taxon, showed unique alleles with several probes and both restriction enzymes. Consequently, both taxa were easily distinguishable from P. fortinii isolates. Whereas EcoRV in combination with seven probes revealed only eight multi-locus haplotypes among 17 ISSR phenotypes, HindIII in combination with 12 probes was able to distinguish all ISSR and/or allozyme phenotypes except two pairs of strains. Strains of P. fortinii showed a high gene diversity, and up to 15 alleles per locus were observed among the 31 strains of P. fortinii. The 12 probes used in combination with HindIII were (partially) sequenced and BLAST searches showed no similarities with known sequences indicating that they probably are non-coding regions of the genome. Enzyme-probe combinations suitable to indicate the genetic structure of P. fortinii and Type 1 populations were easily identified in the present study. This opens up avenues to study communities of the P. fortinii complex exposed to various environments and management intensities, and will help to promote the understanding of these extremely successful organisms.

Bovine spongiform encephalopathy, first identified in the UK in 1986, may have arisen from fe - eding scrapie infected meat-and-bone meal (MBM) to cattle that was produced under sub-optimal conditions. For public health and disease prevention reasons, banning the application of MBM in ru- minant's feeds was necessary. This study utilized a polymerase chain reaction (PCR) assay to iden- tify bovine, porcine, ovine, and chicken MBM in animal diets. Bone fragments from feed samples were separated by chloroform separation. A nucleospin column was used for DNA extraction. Four pairs of primers that targeted at highly conserved regions of mitochondrial DNA (mtDNA) were employed in a PCR procedure to detect bovine, porcine, ovine and chicken DNA sequences. These gene fragments at the targeting region for the four species were 271 bps, 225 bps, 212 bps and 266 bps in size, respectively. In order to confirm the specific amplification of expected products from MBM for each species, the PCR products were digested with restriction enzymes HphI, MnlI, SspI and HindIII, and diffe- rent length polymorphisms were observed. In the sensitivity assays, concentration as low as 1% of MBM in animal feeds could be detected by the PCR procedure. Based on our prevalence survey for commercial MBM in Taiwan, it predominantly consisted of bovine or porcine origins. This molecular approach provides a quick, highly reproducible and sensitive method to detect the MBM in feeds.

Adenovirus is an important cause of respiratory infections in infants and children. Fifty-one serotypes have been identified, and adenovirus type 3 (Ad3) and Ad7 have often been associated with outbreaks of severe respiratory tract infections. Each serotype can be further divided into genome types based on the patterns of digestion of their DNAs with restriction enzymes. DNA restriction analysis was performed with 56 strains of Ad3 and 98 strains of Ad7 by using 12 restriction enzymes recognizing 6 bp ( Bam HI, Bcl I, Bgl I, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal I, Sma I, Xba I, and Xho I). The virus strains were isolated during outbreaks of lower respiratory tract infections in children during an 11-year period from 1990 to 2000 in Seoul, Korea. Among the Ad3 strains, seven genome types were identified; Ad3a and six novel types (Ad3a13, Ad3a14, Ad3a15, Ad3a16, Ad3a17, and Ad3a18). Multiple genome types cocirculated during outbreaks, and some of these were isolated during the 11-year observation period, while others were restricted to particular outbreaks. For Ad7, two genome types, Ad7d and Ad7l, the latter of which is a novel genome type, were identified. A shift in genome types occurred from Ad7d to Ad7l during successive outbreaks. Mortality was 3.6% among children with Ad3 infections and 18% among children infected with either of the Ad7 genome types. In conclusion, the data confirm that Ad3 genome types are more diverse than those of Ad7 and suggest that shifts of genome types may occur during successive outbreaks of Ad3 and Ad7.

To explore the intrafamilial homology and longitudinal stability of colonization by early acquired mutans streptococci, genomic fingerprinting was performed on 254 strains (192 Streptococcus mutans and 62 Streptococcus sobrinus strains) collected from 16 families (16 mother child pairs, seven fathers and four siblings). Genomic DNA was digested by the restriction endonuclease HindIII, followed by gel electrophoresis, Southern blotting, and hybridization with a digoxigenin-labeled 16S rDNA probe, and hybrid detection by enhanced chemiluminescence. One to five ribotypes were identified per person, and between two and nine (median five) within each family. Fourteen of the 16 mother-child pairs showed homology for at least one ribotype (range 1-4). Six of the seven father-child pairs had one ribotype in common. Ten of the 13 longitudinally examined children showed persistence of at least one ribotype over a period of up to 16 yr. The results support the notion of intrafamilial transfer of mutans streptococci, and suggest that colonization of early acquired strains persists into young adulthood.

A single nucleotide polymorphism (SNP) in chromosome Y has been associated with blood pressure. In men, the risk of suffering from cardiovascular diseases, including coronary artery disease, could be influenced by one or more loci on chromosome Y. We genotyped 208 men who had suffered an early episode of myocardial infarction (MI) (≤55 years) and 178 healthy control men for two Y-polymorphisms (a HindIII polymorphism in an alphoid satellite in the centromeric non-recombining region and the −2627 T/C in the SRY gene). Frequencies were compared through a χ2-test. Frequencies for the two polymorphisms did not differ between patients and controls. The alphoid-HindIII polymorphism was not related to blood pressures in our population (HindIII+: diastolic, 80 ± 2; systolic, 129 ± 5. HindIII−: diastolic, 80 ± 2; systolic, 128 ± 3). Seventy-six patients (37%) were hypertensives and had a significantly higher frequency of the HindIII+ allele compared to the normotensive patients (46 and 26%, respectively; P = 0.028). According to our data, the alphoid-HindIII polymorphism in chromosome Y was not associated with differences in blood pressure in men from Asturias (Northern Spain). However, the HindIII+ allele increased the risk of suffering an early episode of MI among hypertensives. © 2003 Wiley-Liss, Inc.

AATCC-3′. When double-stranded, these oligonucleotides have termini that are cohesive with the ends of HindIII-linearized pFOS-1. However, the HindIII site is not regenerated upon insertion of the oligonucleotide duplex into the vector. The central region of the duplex contains recognition sites for two restriction enzymes that leave blunt ends, SwaI and PmlI. The modified vector pFOS-LA was

The Eco VIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the Eco VIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the Eco VIII R-M system was recently acquired by the genome. The 921-bp Eco VIII endonuclease (R · Eco VIII) gene ( ecoVIIIR ) encodes a 307-amino-acid protein with an M r of 35,554. The convergently oriented Eco VIII methyltransferase (M · Eco VIII) gene ( ecoVIIIM ) consists of 912 bp that code for a 304-amino-acid protein with an M r of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of Eco VIII R-M enzymes purified to homogeneity were characterized. R · Eco VIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · Eco VIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N 6 -methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the Hin dIII ( Haemophilus influenzae Rd) and Lla CI ( Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · Eco VIII with M · Hin dIII and M · Lla CI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m 6 N -adenine β-class methyltransferases. The deduced amino acid sequence of R · Eco VIII shows weak homology with its two isoschizomers, R · Hin dIII (26%) and R · Lla CI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · Eco VIII (D 108 X 12 DXK 123 ), as well as in the primary structures of R · Lla CI and R · Hin dIII. Polyclonal antibodies raised against R · Eco VIII did not react with R · Hin dIII, while anti-M · Eco VIII antibodies cross-reacted with M · Lla CI but not with M · Hin dIII. R · Eco VIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · Eco VIII enzyme. The biological implications of this finding are discussed.

Summary A nucleic acid probe for the Herpesvirus anguillae (HVA) Taiwan isolate was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment (1550 bp) generated by digestion of HVA DNA with the restriction enzyme HindIII, and labeled non-radioactively with digoxigenin (DIG). The probe was used to detect the HVA genome from HVA-infected cell cultures and tissue specimens prepared from infected eels, using either dot blot or in situ hybridizations.

Cholesteryl ester transfer protein (CETP) is one of the key proteins in reverse cholesterol transport (RCT). The role of CETP in atherosclerosis remains controversial. In this study we investigated the associations between polymorphisms of CETP (mutations in intron 14 and exon 15, and Taq1B), hepatic lipase (C-514T), lipoprotein lipase ( PvuII and HindIII), and ATP-binding cassette transporter 1 (R219K) loci and longevity in 256 centenarians and 190 healthy younger controls. Although heterozygous CETP deficiency and the B2 allele of the Taq1B polymorphism was consistently associated with higher HDL-C concentrations both in centenarians and controls, the allelic frequencies of those polymorphisms did not differ between the two groups. The allelic frequencies of other gene polymorphisms in RCT were not different between the two groups. Centenarians with lipoprotein lipase P(-/-) genotype had significantly higher HDL-C concentration than those with P(-/+) or with P(+/+), in contrast, there was no such a relationship among controls. In stepwise multiple regression analysis, serum albumin, CETP deficiency and lipoprotein lipase PvuII genotype were independently associated with HDL-C in centenarians. Sex, CETP deficiency, and the Taq1B genotype were also independently associated with HDL-C; however, lipoprotein lipase PvuII genotype had no significant effect on their HDL-C in controls. In conclusion, we observed that CETP deficiency and other gene polymorphisms in RCT have no impact on longevity for Japanese centenarians.

Aims: To investigate the genetic differences among the strains of adenovirus type 8 (Ad8) circulating in Hiroshima city, Japan, and to study their circulation pattern. Methods: One hundred and twenty nine strains of adenovirus type 8 (Ad8) were isolated in Hiroshima City over a 15 year period (1983–97) from patients with keratoconjunctivitis, and analysed with six restriction enzymes—BamHI, HindIII, PstI, SacI, SalI, and SmaI—to investigate possible relations among the isolates and their genetic variability. Seven hypervariable regions of the hexon gene that carry the type specific epitope were also sequenced to investigate the variation among the genome types. Results: Restriction endonuclease analyses yielded three known genome types (Ad8A, 13 samples; Ad8B, seven samples; and Ad8E, 35 samples) and a novel genome type (Ad8I, 74 samples). Ad8A, Ad8B, and Ad8E were closely related, with 96% homology, whereas Ad8I had only 71% homology. Ad8A, Ad8B, and Ad8E shared 91.8% and 96.4% homology with regard to their amino acid and nucleotide sequences, respectively, with the isolate 1127 (accession no X74663). However, when compared with Ad8A, Ad8B, Ad8E, and isolate 1127, Ad8I shared only 62.7% and 69.9% homology with regard to amino acid and nucleotide sequences, respectively. Ad8A, Ad8B, and Ad8E had a unique 31 amino acid deletion in the hypervariable region 1 of the hexon gene, whereas Ad8I had a 33 residue deletion. The Ad8E strain that circulated from 1984 to 1995 was stable among the study population. Ad8I was isolated from an outbreak of epidemic keratoconjunctivitis in 1995 and was also isolated from sporadic cases until 1997. Conclusions: These results confirmed that genetic variability occurs in Ad8 in the microenvironment and revealed the emergence of a new genome type (Ad8I).

Abnormalities in fibrinolysis system is associated with risk of hypertension. In this report, the Alu repeat insertion/deletion (I/D) polymorphism of tissue plasminogen activator (t‐PA) and the Hind III RFLP of plasminogen activator inhibitor‐1 (PAI‐1) genes were investigated in 115 normotensives and 83 patients with hypertension, and their association with anthropometrical data and plasma biochemical parameters were analyzed. There were no significant differences in the gene frequencies of the two candidate genes between normotensives and hypertensives, respectively. Our results indicate lack of associations between the two polymorphisms in t‐PA and PAI‐1 genes and risk of hypertension in the population under study. However, the Hind III RFLP of PAI‐1 gene was significantly associated with plasma glucose level, suggesting its role in glucose metabolism. It needs to be tested whether this RFLP of PAI‐1 gene is associated with insulin resistance syndrome or non‐insulin dependent diabetes mellitus (NIDDM) i...

The objectives of the research were to detect genetic variations on ND-5 region of mtDNA of Benggala and Madura cattles, and to compare the genetic diversity within or between Madura and Benggala cattle. Genetic variations and its effects on phenotype characters have been studied largely in dairy cattles, but not for beef cattles, especially for Indonesian local cattles. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was used to detect polymorphism on ND-5 region of mitochondrial DNA. Polymorphisms were found on ND-5 mitochondrial DNA fragment using HindIII restriction enzyme. This variation were likely due to lost of HindIII restriction site on ND-5 mithocondrial DNA fragment.© 2003 Jurusan Biologi FMIPA UNS SurakartaKey words: ND-5 mitochondrial DNA, genetic variation, PCR-RFLP.

One hundred thirty-two strains of adenovirus type 7 were isolated in Hiroshima City during the period 1995–1999. About 80% of the viruses were isolated from patients with respiratory illness, and about 70% were isolated from children younger than 6 years old. DNA restriction analysis was performed on 58 of the 132 isolates. Two genome types corresponding to adenovirus 7d and adenovirus 7h were recognized using BamHI. By using an additional 10 restriction endonucleases (BclI, BglI, BglII, BstEII, HindIII, HpaI, SmaI, PstI, PvuII, and SacI), the 58 isolates were classified finally into five genome types according to Wadell and coworkers' nomenclature system: adenovirus 7d2 (54 isolates), adenovirus 7d3 (one isolate), adenovirus 7d4 (one isolate), adenovirus 7d5 (one isolate), and adenovirus 7h (one isolate), among which adenovirus 7d3, 7d4, and 7d5 were new genome types. Our results indicate that the predominant genome type of adenovirus 7 that circulated in Hiroshima City during the period May 1995–November 1999 was adenovirus 7d2. J. Med. Virol. 69:215–219, 2003. © 2003 Wiley-Liss, Inc.

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763 T and C. mobile DSM 4849 T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766 T and C. piscicola NCDO 2762 T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA Ile and tRNA Ala genes. The M-ISR included one tRNA Ala gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with Hin fI, Taq I, and Hin dIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.

In Vietnam, the carrier rate for beta-thalassemia varies from 1.5% to 25% depending on the ethnic groups of the population. The molecular basis of beta-thalassemia in South Vietnam was studied in 50 unrelated beta-thalassemia patients. Of these, 31 had beta-thalassemia/Hb E, 18 were homozygous for beta-thalassemia, and 1 carried the beta-thalassemia trait. The majority of the patients were Kinh, four were Chinese, and two were Kinh-Chinese. All had severe anemia and received blood transfusions regularly, every 1-3 months. Hepatosplenomegaly was found in all patients, and splenectomy had been done in six patients. Normal alpha-globin genotype (alpha alpha/alpha alpha) was found in all subjects. Reverse dot-blot hybridization using oligonucleotide probes specific for Southeast Asian mutations can detect beta-thalassemia in 60 chromosomes in addition to 31 chromosomes with beta(E) mutation. Excluding the beta(E) gene, six previously reported Thai and Chinese beta-thalassemia mutations were found, including codons 41/42 (-TCTT) 35.3%, codon 17 (A-->T) 25.0%, -28 (A-->G) 7.3%, codons 71/72 (+A) 7.3%, IVS-II nt 654 (C-->T) 7.3%, and IVS-I nt 1 (G-->T) 6.0%. The Vietnamese frameshift mutation at codon 95 (+A) was detected by ARMS in seven chromosomes (10.3%). DNA polymorphism of the beta-globin gene cluster carrying the codon 95 mutation was - + - - - - - + for (G)gamma/XmnI, epsilon/HincII, (G)gamma/HindIII, (A)gamma/HindIII, psi beta/HincII, 3' psi beta/HincII, beta/AvaII, and 3'beta/BamHI, respectively. The remaining mutation detected by the gap PCR was a large deletion known as the Chinese (G)gamma((A)gamma delta beta)(0)-thalassemia. The two most common mutations were the frameshift at codons 41/42 (-TCTT) and the nonsense mutation in codon 17 (A-->T). Thus beta-thalassemia mutations in South Vietnam is similar to the previous report from the North, although at different frequencies. This result will help to establish a center for prenatal diagnosis and for prevention and control of thalassemia in Vietnam.

The prevalence of Actinobacillus actinomycetemcomitans isolates was examined in a rural population of southern Thailand. Sixty individuals aged 30-39 and 50-59 years were randomly selected from a group of 363 persons, living in four villages, who had been clinically examined previously. A subgingival plaque sample was taken with a curette from the mesial aspect of the two upper and lower first molars. Each sample was dispersed in 3.3 ml of VMGA III transport medium and spread onto Trypticase Soy Broth with Bacitracin and Vancomycin (TSBV)-agar plates on the same day. After incubation in 10% CO 2 for 5 days the plates were examined for typical A. actinomycetemcomitans colonies which were tested for catalase activity. Each strain was further tested for biochemical characteristics, serotyped against serotype-specific antisera a-e and ribotyped after DNA digestion using the restriction endonucleases HindIII and EcoRI. For 53 (88%) of the 60 individuals, A. actinomycetemcomitans was present in at least one subgingival sample, which is considerably higher than the prevalence in Western European adults. In 11 individuals, two or three different strains were found. Serotypes a and c were the most prevalent, and serotype b was found only once among 46 tested isolates. Eleven ribotypes were found among the 46 strains. While the same ribotype could be found among individuals of the same village, no ribotype of A. actinomycetemcomitans was unique for individuals of any one village. The study demonstrated a high prevalence of A. actinomycetemcomitans among adults of the rural population of southern Thailand and indicates that this species is present as part of the resident oral flora in this population.

Ukkola O, Tremblay A, Bouchard C (Louisiana State University, Baton Rouge, LA, USA; University of Oulu, Oulu, Finland; and Laval University, Ste-Foy, Quebec, Canada). Lipoprotein lipase polymorphisms and responses to long-term overfeeding. J Intern Med 2002; 251: 429–436. Objectives. The role of the lipoprotein lipase (LPL) gene Hind III, S447X, Bam HI and Pvu II polymorphisms on body composition and lipid and lipoprotein changes in response to long-term overfeeding was studied. Subjects. Twelve pairs of male monozygotic twins ate a 4.2 MJ day−1 energy surplus, 6 days a week, during a period of 100 days. Results. Overfeeding induced a decrease in high-density lipoprotein 2 cholesterol (HDL2-C) and HDL2-C to HDL3-C ratio in the H2H2 (n = 12) subjects of the LPL Hind III polymorphism. In contrast, the H1H1/H1H2 (n = 12) subjects experienced increases both in the HDL2-C and HDL2-C to HDL3-C ratio (P = 0.009 and 0.007, respectively, for differences in percentage changes between H2H2 and H1H1/H1H2). In addition, the H2H2 genotype was associated with higher levels of very-low-density lipoprotein triglyceride (VLDL-TG) (P

Four hundred and twenty-two beef cattle of two different breeds (purebred Hereford and composite multibreed) were characterized by polymerase chain reaction-restriction fragment length polymorphism, using the restriction enzymes ApaI, AvaII, HindIII, PstI, SpeI, SspI and TaqI in two regions (the D-loop and the ND-5 gene) of mitochondrial DNA. The association between molecular haplotypes and records on calving rate, defined as the mean number of live calves born per year over 4 years, were examined by analysis of variance. A significant association was found between calving rate and mitochondrial polymorphisms in both breeds. This may have implications for genetically improving cow fertility.

Higher blood pressure (BP) in males compared with females is well documented and is thought to be influenced in part by the Y chromosome. To examine whether there is an association between BP and a polymorphic Hin dIII biallelic marker in the nonrecombining region of the Y chromosome, we genotyped 155 males from a Polish study group and 762 males from a Scottish study group. We also tested for possible interaction between the Y chromosome and a mutation in the steroidogenic factor binding site of the aldosterone synthase gene by genotyping the same group from Scotland. There was no significant difference in age or body mass index between 2 Y chromosome genotypes in both study groups. Men with the Hin dIII(+) genotype had significantly higher systolic and diastolic pressures than those with the Hin dIII(−) genotype in both the Polish and Scottish studies. This difference between the genotypes was 5.27 mm Hg ( P =0.0014) and 3.14 mm Hg ( P =0.0005) for adjusted systolic BP and 2.6 mm Hg ( P =0.0045) and 1.44 mm Hg ( P =0.0084) for adjusted diastolic BP in the Polish and the Scottish studied, respectively. On binary logistic regression analysis, males with the Hin dIII(+)/TT SF1 genotype combination had an odds ratio for elevated BP of 3.92 (CI 1.21 to 12.68, P =0.023). Our results indicate that the Y chromosome harbors a locus or loci that contribute to BP variation in hypertensive and normotensive men. The polymorphism in the aldosterone synthase gene may interact with the Y chromosome to increase the odds of an individual’s developing higher BP.

The high molecular weight glutenin subunit (HMW-GS) composition of a collection of 107 Argentinean bread wheat cultivars was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Allelic variation at the Glu-1 loci was identified and its frequency calculated. Eleven alleles were detected, three encoded at the Glu-A1 locus, six at the Glu-B1 locus and two at the Glu-D1 locus. A low frequency of the null allele at the Glu-A1 locus and a high frequency of subunits 5+10 at the Glu-D1 locus were observed. Reversed phase-high performance liquid chromatography (RP-HPLC) analysis was used to further characterise HMW-GS. Two different types of Bx subunit 8 (named subunits 8 and 8*) were detected, the latter having shorter elution time. Subunit 8* was not identifiable by SDS-PAGE. According to quantification by RP-HPLC analysis, two groups of subunit 7 were observed. One group, with a relatively high proportion of subunit 7 (approximately 39% of the total amount of HMW-GS) appeared in cultivars with allele 7+8* at the Glu-B1 locus; a second group of subunit 7 (around 24% of the total amount of HMW-GS), was found in alleles 7+8, 7+8* and 7+9. Restriction fragment length polymorphisms (RFLP) analyses of HMW-GS genes were also carried out after digestion of genomic DNA with HindIII and TaqI restriction enzymes. The relationship between DNA fragment size and glutenin subunits, as estimated by electrophoretic mobility in SDS-PAGE, was also examined. The restriction enzyme TaqI demonstrated to be a useful tool to detect homozygous plants in selection programs against the Glu-A1 null allele.