Search

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.

Background It is known that patients with sickle cell disease (SCD) present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises and also during the steady state of the disease. We determined whether the presence of the factor prothrombin gene G20210A variant, factor V gene G1691A mutation (factor V Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms may be risk factors for vascular complications in individuals with SCD. Methods The study involved 150 patients with sickle cell anemia and 150 healthy controls of Central India. Genotyping of three thrombophilic mutations was carried out by PCR-RFLP methods using MnlI, Hind III, and Hinf I, respectively, for factor V Leiden, prothrombin, and MTHFR mutations. Results Patients with SCD had significantly higher prevalence of mutant variants of MTHFR gene (28.0% heterozygotes and 14.6% homozygotes) and FVL gene (14.6% heterozygotes) as compared to normal/control individuals, but complete absence of mutant variants of prothrombin gene. The patients with SCD having mutant variants of MTHFR and FVL genes showed higher incidence of pain in chest, abdomen, and bone joints along with early age of onset of clinical manifestations as well as frequent dependence on blood transfusion than those patients with SCD having wild variants of these thrombotic genes. As compared to control subjects, SCD individuals having mutant variants of FVL and MTHFR genes had significant association with higher levels of prothrombin fragment (F1+2), D-dimer, thrombin-antithrombin (TAT), and lower level of protein C. Conclusion MTHFR C677T and FVL G1691A polymorphisms may be risk factors for increased vascular complications in patient with SCD.

PIRA-PCR (primer-introduced restriction analysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the b2-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A HindIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I( Tsp4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both HindIII and TaaI. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7‐8 h while the conventional method required 5‐7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.

Thirty penicillinase-producing Neisseria gonorrhoeae (PPNG) strains, 26 representing 9 serovars of the protein IB and 4 representing 2 serovars of the protein IA serogroup, had 10 different patterns when investigated by the restriction enzyme (RE) technique with Hind III enzyme. One RE pattern dominated, representing 11 strains with 6 different serovars of both the protein IA and IB serogroup. Identical serovars and RE patterns were found among 7 patients (3, 2 and 2, respectively) with positive epidemiology. The majority of the strains (24/30) were resistant to, or showed decreased sensitivity to, doxycycline with minimum inhibitory concentrations between 1 and 8 mg/l, consistent with the Far East origin. All the 30 PPNG strains showed by agarose gel electrophoresis to harbour a 2.8 Mdalton, a 4.4 Mdalton, as well as a 24.5 Mdalton plasmid, whereas the two non-PPNG strains had only the 2.8 Mdalton plasmid.

Summary We identified the HindIII restriction site polymorphism of K-casein in cattle reported by Pinder et al. (Animal Genetics 22, 11, 1991) and found an additonal polymorphism (RsaI) in cattle and bison. The HindIII and RsaI restriction sites were mapped and three haplotypes (alleles) were identified. Preliminary screening of 39 cattle and 71 bison revealed one allele restricted to cattle, one restricted to bison, and one shared by the species. No fixed allelic differences were observed among cattle breeds or among bison herds or subspecies.

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.

Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.

Two bovine populations, a Holstein-Friesian dairy stock and a synthetic (Baladi X Hereford X Simmental X Charolais) beef stock, were screened for restriction fragment length polymorphisms (RFLPs) at the growth hormone and prolactin genes. Most RFLPs at the growth hormone gene are apparently the consequence of an insertion/deletion event which was localized to a region downstream of the structural gene. The restriction map for the genomic region including the growth hormone gene was extended. Two HindIII RFLPs at the growth hormone locus, as well as several RFLPs at the prolactin gene, seemed to be the consequence of a series of point mutations. The results are discussed in terms of the possibility that minor genomic variability underlies quantitative genetic variation.

The human colonic adenocarcinoma cell line HT-29 does not normally express HLA class II molecules. By restriction fragment length polymorphism (RFLP) analysis of DNA with a DR beta-probe, we analysed the genomic DR beta polymorphism of this cell-line, and compared it with the RFLP patterns seen in DNA from a reference panel of different DR homozygous and heterozygous cells. The HT-29 cell-line expressed DR beta fragments similar to the sum of the fragments expressed by DR4 and DR7 homozygous cells. The DR4 and DR7 haplotypes of HT-29 was further confirmed by comparing the RFLP patterns of four DR4/7 heterozygotes with that of HT-29. Furthermore, the HT-29 cell line expressed a Hind III 9.3 kb fragment previously found to be strongly associated to DRw53. Following treatment with gamma-interferon, the HT-29 cells could be induced to express class II molecules. Serological typing revealed the presence of the DR4, DR7 and DRw53 antigenic determinants.

Summary In order to evaluate the feasibility of first trimester prenatal diagnosis of β-thalassaemia by restriction fragment length polymorphism (RFLP) in Campania, one of the most affected regions in Southern Italy, DNA polymorphism analysis was performed on 40 unrelated patients, affected with homozygous β-thalassaemia, and on their parents. Frequency of the presence of the Hinc II e, Hind III Gγ and Aγ, Hinc II SPSβ and SPSβ, Ava II SPSβ, Ava II β and Bam HI 3′β sites have been determined in the βA and βthat chromosome samples. In 31 families (over 75%). RFLPs enabled tracing the β-thalassaemia mutations in both father and mother (100% diagnosis). In the remaining nine families, RFLPs enabled tracing only one of the two mutations (50% diagnosis) because the other parent was found to be homozygous in all the analysed polymorphic sites. Restriction haplotypes, assembled on the basis of linkage analysis, were most heterogeneous, hence a wide heterogeneity of mutations is expected.

To study the association of renin gene polymorphism with essential hypertension in the Chinese population, 86 hypertensive and 107 normotensive subjects were enrolled from an epidemiologic survey. Leukocyte DNA was extracted and digested with Hind III and Bgl I restriction enzymes. Southern hybridization was done with digoxigenin-incorporated renin gene probes generated by polymerase chain reaction. The restriction fragments were detected by anti-digoxigenin antibody and enzyme methods. Two Hind III polymorphysms of the renin gene (8.7 kb and 6.2 kb) were identified. The allele frequences were 129(75%) and 43(25%), respectively, in hypertensives; they were 139(65%) and 75(35%), respectively, in normotensives (chi2 = 4.074, p = 0.044). The genotypes of 8.7/8.7,8.7/6.2 and 6.2/6.2 were significantly different between hypertensives and normotensives, being 45(52%), 39(45%), 2(3%) and 48(45%), 43(40%), and 16(15%), respectively (chi2 = 9.002, p = 0.11). The Bgl I polymorphism did not show a difference between hypertensives and normotensives. Thus, we conclude that the renin gene Hind III polymorphysm is associated with hypertension in this Chinese population.

The usefulness of intragenic restriction fragment length polymorphisms (RFLPs) for BclI, HindIII and XbaI, adapted for polymerase chain reaction (PCR), was tested for the detection of haemophilia A carrier status in the consultant of a family in which only haematological information was available on the inheritance of the trait. Hair follicles were used as the non-invasive source of DNA. The mother was found to be homozygous for BclI and heterozygous for HindIII sites, whereas her status as regards informativeness could not be established for XbaI. On the basis of HindIII RFLP, the daughter was found to be a carrier of the haemophilia trait. This was confirmed by sequencing the amplified intron 19 of the mother and the daughter. The RFLP for XbaI did not appear to be suitable for carrier detection using PCR due to the difficulty of establishing homozygosity or heterozygosity from the results of digestion of the amplified product.

Summary Rearrangements within the major breakpoint cluster region (M-BCR) were demonstrated by Southern blot analysis for 74 of 80 patients with Philadelphia (Ph1) positive chronic granulocytic leukaemia (CGL) and for one and three patients with Ph1 negative CGL. Multiple restriction enzyme digestion permitted the breakpoint within the M-BCR to be localized relative to the central Hind III site for 71 patients. 62% had 5′ rearrangements and 38% had 3′ rearrangements. Those with 3′ rearrangements had significantly higher platelet counts at presentation than those with 5′ rearrangements (P < 0.001). We examined factors (such as patient selection criteria and retrospective analysis) which may have influenced previous reports of the prognostic value of the breakpoint location within M-BCR. For patients diagnosed after November 1988 (prospective cases), no significant difference was observed for the duration of chronic phase for patients with 5′ or 3′ rearrangements (P= 0.83, Logrank test). We conclude that, although higher presentation platelet counts were associated with 3′ rearrangements, the location of the breakpoint within the M-BCR appears to be of limited prognostic value.

Background and Objective: The plasminogen activating system is a protease/inhibitor system central to extracellular matrix remodeling with a suggested role in periodontal disease pathology. A few studies have reported polymorphisms in the genes of plasminogen activator inhibitors to be associated with periodontal disease severity. Two gene polymorphisms – a BamHI restriction fragment length polymorphism in the urokinase plasminogen activator gene (uPA) and a HindIII restriction fragment length polymorphism in the plasminogen activator inhibitor type 1 gene (PAI-1) – have been associated with conditions having a vascular component, and our objective was to assess the association of these gene polymorphisms with alveolar bone loss in chronic periodontal disease of adults. Material and Methods: Genotype was determined by polymerase chain reaction amplification of whole blood, pertinent histories were obtained by interview, and alveolar bone loss was assessed from current radiographs. Results: In 77 elderly patients with a normal distribution of alveolar bone loss, we demonstrated a significant association between levels of alveolar bone loss and these polymorphisms in the uPA and PAI-1 genes. Controlling for the contributions of smoking or diabetes to periodontal bone loss, estimated odds ratios for predicting lower levels of alveolar bone loss, associated with a greater degree of periodontal health, were strongest when defined by the concurrent presence of a homozygous urokinase plasminogen activator genotype and the nuclease-sensitive plasminogen activator inhibitor type 1 (HindIII) allele (odds ratio = 2.6; 95% confidence interval: 5.8–1.3). Conclusion: The urokinase plasminogen activator (BamHI) and plasminogen activator inhibitor type 1 (HindIII) genotypes may serve as useful markers for heritability of bone loss associated with periodontal disease.

Haemophilia A is an X-linked, recessively inherited bleeding disorder of varying severity, which results from the deficiency of procoagulant factor VIII f(8). Linkage diagnosis using polymorphic markers in the f8 gene is widely used to detect carriers. The objective of this study was to verify the informativeness of three polymorphic markers in the Brazilian population, to evaluate the usefulness of such markers in carrier detection procedures. Sixty-three unrelated healthy volunteers and 10 haemophilic families were studied. Two microsatellite repeats and one HindIII RFLP markers were used. Carrier and non-carrier status could be determined in 80% of females investigated. Intron 13 markers presented the highest heterozygosity rate (79%) followed by intron 22 (68%) and intron 19 (57%). When all three markers were used together, linkage analysis informativeness increased significantly. We conclude that these markers are suitable for carrier detection in the Brazilian population and we recommend their use in combination to maximize diagnostic efficiency.

The phenotypic stability of the recombinant plasmid pAT9 (11.5 MD), which contained the cloned α-amylase gene from B. stearothermophilus, was studied in batch and continuous culture. Irrespective of the type of culture (batch or continuous; or using glucose or maltose as carbon source), deletion plasmids of the phenotype KmrAmy− the same size (7.3 MD) emerged with time. DNA sequencing analysis and Southern hybridization of a region adjacent to both ends of the pAT9 HindIII fragment containing the α-amylase gene showed that almost the entire part of the HindIII fragment was lost. Homology between fragments contiguous to the pAT9 HindIII terminals allowed exclusion of a fragment that did not encode the α-amylase gene and a new recombinant plasmid, pATHP9 (KmrAmy+; 7.5 MD), was constructed which had enhanced phenotypic stability in B. stearothermophilus.

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.

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.

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.

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.

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.

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

EcoRI and HindIII restriction fragment length polymorphism (RFLP) profiles using 2 random DNA probes, named 104 and 106, were generated for 85 B. thuringiensis strains. These include 80 serovars, 4 intra-serovar strains: kurstaki HD-1, dendrolimus, tenebrionis and sandiego, and a non-serotypeable strain B. thuringiensis var. wuhanensis. A total of 47 EcoRI and 65 HindIII restriction patterns were generated when hybridization results from both probes were combined. Seventy-seven B. thuringiensis strains showed distinctive hybridization profiles. The dendrogram resulting from the numerical analysis of the distance matrix revealed fourteen distinct phylogenetic groups at the 96% banding patterns similarity. The intra-serovar strains showed higher similarity with their respective type serovars. However, different serovars from a common H-serotype did not always cluster in the same phylogenetic group. Alternatively, several mosquitocidal serovars clustered in a single phylogenetic group. The correlation between serotyping and banding pattern similarity is discussed.

Twelve strains of adenovirus serotype 19, isolated from cases of epidemic keratoconjunctivitis in Japan in 1992, 1993, 1997, and 1998, were analyzed by DNA restriction analysis, using restriction endonucleases BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, SacI, SalI, SmaI, and XhoI. Among these 11 restriction endonucleases, EcoRI, PstI, SacI, and SmaI were discriminative enzymes, showing restriction patterns different from those reported previously for the prototype and the variant 19a. This new genome type was isolated in 1997 and 1998, when an increase of epidemic keratoconjunctivitis cases caused by adenovirus serotype 19 was observed for both sporadic and nosocomial infections. Strains from 1992 and 1993 showed restriction patterns similar to those of the worldwide reported variant 19a for all enzymes used. The changes detected in strains from 1997 and 1998 could be the reason for the recent epidemic.

Osteoporosis is a common disorder with a strong genetic component. Our aim was to evaluate the correlation of the HindIII osteocalcin gene polymorphism to bone mineral density (BMD) and their relationship to osteoporosis. We determined the HindIII osteocalcin gene polymorphism using polymerase chain reaction (PCR)‐based restriction analysis in postmenopausal Chinese women in Taiwan. The osteocalcin gene polymorphism was detected by the restriction enzyme HindIII, where the H allele indicated the absence of the cuttable site and the h allele indicated its presence. We then related the genotypes to BMD and occurrence of osteoporosis in these women. The allelic frequencies for postmenopausal Chinese women in Taiwan were 64% for h and 36% for H in HindIII restriction fragment length polymorphisms. The prevalence of each genotype in the study population was 37.7% hh, 52.6% Hh, and 9.7% HH. The subjects with genotype hh had the greatest BMD at the lumbar spine and the femoral neck, and those with HH had the smallest BMD at the femoral neck, but these differences did not reach statistical significance. The HindIII osteocalcin genotype showed a significant effect on the prevalence of osteoporosis in the subjects at the femoral neck, that is, women with genotype HH had a 6.4 times greater risk for osteoporosis (P < 0.05), and those with genotype Hh had a 1.2 times greater risk than women with genotype hh. In conclusion, the HindIII osteocalcin gene polymorphism is associated with reduced BMD and predisposes women to osteoporosis at the femoral neck. J. Clin. Lab. Anal. 15:251–255, 2001. © 2001 Wiley‐Liss, Inc.

Solid-Phase DNA restriction digest reactors have been developed consisting of silica particles modified with a covalently tethered restriction enzyme. This solid-phase restriction reactor enables digestion and separation of minute quantities of DNA with minimal reagent consumption. In this study, the restriction enzymes, HaeIII, PstI, and HindIII, were successfully immobilized via glutaraldehyde linkages to porous silica micro-particles. Studies were carried out to examine the impact of immobilization on enzymatic activity. Digestions of ϕX174-RF DNA phage and SV40 viral DNA were performed with the immobilized enzymes by placing the silica particles in solution with the target DNA with digestion times of 120 min and 240 min respectively. The digests were analyzed off-line using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Timed studies were performed to establish optimal conditions for complete digestion. Digests utilizing immobilized HaeIII and HindIII were similar in composition to homogeneous, free solution digests. PstI showed no evidence of activity upon immobilization. The immobilized restriction enzymes could also be used for multiple rounds of digestion; however, longer incubation times were required for successive runs probably due to partial denaturation of the restriction enzyme. Digests also were prepared and isolated by use of a simple micro-spin column consisting of a layer of immobilized enzyme-coated silica on a molecular weight cut-off filter. Using this approach, digestion times were comparable to solution digests as previously mentioned; however, enzyme reuse and reaction product isolation was facilitated.

Little is known about the heterozygous frequency of factor VIII gene markers in the Asian Indian population. The objective of this study was to establish the heterozygous frequency of polymorphic markers within and flanking the factor VIII gene in Indians and identify those most informative for carrier screening and prenatal diagnosis. Factor VIII gene polymorphism analysis at intragenic and extragenic sites was carried out by the polymerase chain reaction (PCR) method and Southern blot procedure. Sixty-three Asian Indian haemophiliacs and their families were screened. A control group of 150 women from nonhaemophilic families were screened for two markers, HindIII and BclI. Among the intragenic markers studied, the HindIII restriction fragment length polymorphism (RFLP) showed the highest heterozygous frequency (0.52) followed by the intron 13 (0.47) and intron 22 (0.44) short tandem repeats (STRs). Among extragenic markers, TaqI had the highest heterozygous frequency (0.75) followed by BglII (0.54). The intron 22 inversion mutation was observed in eight (40%) of 20 severe cases. In the population studied the most diagnostic polymorphisms were the intragenic markers, intron 22 (70%) STR followed by the intron 13 (52%) STR and HindIII (52%) RFLP, and the TaqI (50%) extragenic marker. Application of HindIII, BclI and the intron 22 dinucleotide repeat combined were diagnostic in 87.2% of haemophilia A families studied.

Genome analysis was carried out on adenovirus strains isolated from patients with acute follicular conjunctivitis in the city of Sao Paulo, Brazil. Eighteen conjunctival scrapings, collected between December 1993 and March 1994, were analyzed by two methods: a combination of polymerase chain reaction with restriction fragment length polymorphism and viral DNA restriction analysis, carried out using 10 restriction endonucleases: BamHI, BglI, BglII, HindIII, KpnI, SacI, SalI, SmaI, XbaI, and XhoI. Among 11 adenovirus detected by cell culture isolation, nine were Ad8, and two were Ad7. By restriction analysis the Ad8 isolates were typed as two new variants–Ad8/D11 (seven of nine samples) and Ad8/D12 (two of nine samples). Ad7 isolates were identified as a subtype of the widespread genome type Ad7b and the virulent type Ad7h, a predominant genome type circulating in Argentina, Chile, and Uruguay but absent in Brazil until 1991. J. Med. Virol. 61:143–149, 2000. © 2000 Wiley-Liss, Inc.

In the present study, the potential involvement of Streptococcus oralis in the aetiology of recurrent aphthous stomatitis (RAS) was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. PCR was carried out using a primer pair that targets the D-alanine:D-alanine ligase gene and detects DNA from both S. oralis and the closely related species Streptococcus mitis. Discrimination between these two species was achieved by digestion of PCR products with the restriction endonucleases HaeIII and HindIII, which both give distinct restriction profiles for each species. S. oralis DNA was detected in 8 of 28 (29%) RAS samples, 10 of 20 (50%) OLP samples and 6 of 13 (46%) normal samples. These results suggest that S. oralis is not of primary aetiological significance in RAS.

Adenovirus type 3 (Ad3) was the most frequently isolated serotype from patients with conjunctivitis during the period from 1989 to 1991 in Japan. All Ad3 strains isolated in 1990 had an identical genotype, Ad3f. However, in 1992, the predominant serotype was replaced by Ad4. The genome type was examined to determine whether genetic changes existed in Ad3 isolates in 1992. Ad3 isolated from 55 patients with acute conjunctivitis during the period from June 1992 to February 1993 in Japan was assessed by genome typing with restriction endonucleases BamH I, Bgl II, Hind III, and Sma I recognizing 6-base-pair sequences. The emergence of four new genotypes of Ad3 was identified; one with a new Bam HI site (one isolate), one with a new Bgl II site (two isolates), one with a new Hind III site (three isolates) and one with a new Sma I site (two isolates). This study demonstrates that the emergence of a new genotype of Ad3 may contribute to the replacement of the predominant serotype associated with adenovirus conjunctivitis in Japan.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.

Elevated hematocrits, which are found in many high-altitude populations, increase the oxygen-carrying capacity of blood and may represent an adaptation to hypoxic environments. However, as high hematocrit increases blood viscosity, which in turn is associated with hypertension and heart disease, it may be advantageous for high-altitude populations to limit other factors that contribute to increased blood viscosity. One such factor is the plasma concentration of the coagulation protein fibrinogen. Several common polymorphisms in the β-fibrinogen gene have been identified that affect fibrinogen concentrations. We determined the allele frequencies of three of these polymorphisms (G/A -455 (HaeIII), C/T -148 (HindIII), and G/A + 448 (MnlI)) in sample groups drawn from three populations: Quechua-speaking natives living at over 3,200 m in the Peruvian Andes, North American natives (Na-Dene) from coastal British Columbia, and Caucasian North Americans. The frequencies of the alleles previously shown to be associated with increased fibrinogen levels were so low in the Quechuas that their presence could be accounted for solely by genetic admixture with Caucasians. Frequencies in the Na-Dene, a Native American group unrelated to the Quechua, were not significantly different from those in Caucasians.

In about half of the severe haemophilia A cases, the disease is caused by an inversion that split the F.VIII gene into two parts separated by ≈ 300–400 kb. Herein, we show that in the Turkish population this inversion mutation accounts for 29% of 141 haemophilia A cases and 42% of severe cases. Most of the inversions are of the distal type (72%) whereas nine were of the proximal type (28%). Haplotype analysis using 4 markers in the F.VIII gene did not reveal a single haplotype associated with the inversion. However, the pre- valence of one haplotype: HindIII (–) – Int13 (CA)20– Int22 (CA + CT)26–XbaI (–) is higher in the inversion patients. Since founder effect is excluded for the inversion patients, our results suggest that some as yet unknown factor(s) may make these alleles more prone for the inversion. However, a bias due to the low number of studied cases cannot be excluded.

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.

The restriction site mutation (RSM) assay was developed in this laboratory for the detection of point mutations that occur within restriction endonuclease recognition sequences in the genomic DNA of the rat. Mutations were detected and identified in a number of tissues from N-methyl-N-nitrosourea (MNU)-treated rats. Resistant restriction enzyme products were detected in 5 of the 13 restriction endonuclease recognition sequences tested (NcoI, BslI, CfoI, DdeI, and HindIII). These mutations were detected in the p53 tumor suppressor gene and the H-ras protooncogene. No resistant RSM products were detected in any of the samples taken from untreated animals. The MNU-induced mutations were identified as G to A and A to G transitions. Our results describe the first successful application of the RSM assay in detecting induced mutations in the rat and highlight the usefulness of the RSM assay in the analysis of mutagen-induced base changes without the requirement for selection of a mutant phenotype. Given the increasing use of the rat as an animal model in genotoxicity studies, the development of such tests is essential for future genotoxicity investigations.

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8 kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clinical specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraffin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases Hhal and Sacl. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection.

The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other. The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members. Seven patients with moderate to severe periodontitis were selected. These probands, their spouses and 14 of their children were investigated. The presence of Pr. intermedia and Pr. nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa. Differentiation of Pr. intermedia and Pr. nigrescens was performed by enzyme electrophoretic mobility. From all 7 patients, as well as 4 spouses and 3 of the children, Pr. intermedia could be isolated. Pr. nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5-10 yr. In the 8 children aged 0-4 yr both species were seldom isolated. These data are in accordance with earlier findings that Pr. intermedia is associated with periodontitis and Pr. nigrescens with a relatively healthy periodontal condition. Ribotyping of bacteria was performed by hybridization of HindIII restriction endonuclease digests of chromosomal DNA with ribosomal DNA. Isolates from unrelated individuals always had distinct ribotypes. Indistinguishable ribotypes of Pr. intermedia and Pr. nigrescens were found both among married couples and among parents and children. This indicates that intrafamilial transmission of Pr. intermedia and Pr. nigrescens is possible both between adults and between parents and children.

In insulin-dependent (Type 1) diabetes mellitus (IDDM) the development of nephropathy is partly due to genetic susceptibility. Previously one study has demonstrated a relationship between a HindIII restriction polymorphism of the collagen IV alpha 1-chain gene and diabetic nephropathy. The aim of the present study was to evaluate such as association in a case-control study including 207 Danish IDDM patients: 116 with nephropathy (urinary albumin excretion rate (AER) > 300 mg 24 h-1) and 91 without nephropathy (AER < 30 mg 24 h-1). Using genomic DNA, HindIII restriction fragment length analysis revealed a bi allele polymorphism visualized by southern hybridization with a cDNA probe recognizing the collagen IV alpha 1-chain gene. No differences in genotype frequencies or allele frequencies were demonstrated comparing patients with and without nephropathy: p = 0.39 and p = 0.96, respectively. Neither were there any difference in genotype frequencies or allele frequencies when the patients were stratified according to the presence of proliferative retinopathy: p = 0.44 and p = 0.84, respectively. Pooling the diabetic groups revealed genotype frequencies and allele frequencies comparable to those found in 57 healthy unrelated Danish individuals. We conclude that in a Danish IDDM population a HindIII restriction polymorphism of the collagen IV alpha 1-chain gene is not associated with diabetic nephropathy, diabetic retinopathy or with diabetes per se.

Nine Borrelia burgdorferi sensu lato isolated in Korea were subjected to genomic hybridization using 16S rRNA gene probe and specific restriction patterns (HindIII and EcoRV) led these nine Borrelia into five subtypes. The evolutionary relationships of the five isolates corresponding to five RFLP groups were measured through the sequence determination of 16S rRNA gene and phylogenetic analysis. The isolates 935T (group I), 934U and 17Y (Group IIa, IIb) were well clustered with B. garinii and B. afzelii. 5MT and 9MT strains (Group IIIa and Group IIIb) formed a common branch shared with B. afzelii cluster although the evolutionary distance was rather long. So, most of B. burgdorferi sensu lato in Korea was B. afzelii or B. afzelii-related group and some minor group such as B. garinii also existed.

We studied the molecular alterations of IL-6R, lck and c-myc genes in the tumour cells of 50 patients with multiple myeloma (MM) and 20 patients with monoclonal gammopathies of undetermined significance (MGUS). Southern blot analysis revealed a 5.3 kb IL-6R amplified band by digestion with EcoRI and HindIII in three MGUS patients, but no IL-6R gene alteration was found in MM patients. BamHI digestion revealed a 6.2 kb rearranged band of the lck gene in two MGUS patients with IL-6R amplification. In one MGUS patient we detected a rearrangement upstream of the lck coding region. Myc rearrangement was observed in three MM and two MGUS patients who showed coexistent lck rearrangement and IL-6R amplification. These molecular alterations were detected in the MGUS patients with an IgA monoclonal component, who showed a rapid progression into aggressive MM. Myc rearrangement seems to be associated with a small group of a clinically aggressive MM at diagnosis, secreting IgA or k light chains. Multiple rearrangements and/or molecular alterations are likely to occur at the time of MGUS-IgA transformation into aggressive MM and myc rearrangement may promote a positive pressure for transformation and progression. MM tumourigenesis remains a heterogenous multistep process involving the deregulation of many genes and gene products.

Summary Four different clones, PG2, PG3, PG5 and PG6, were isolated by screening a cosmid library of an inbred homozygous SLA typed pig with a human MHC class I cDNA probe (Hertner 1991). These clones were mapped with four different restriction endonucleases (EcoRI, BamHI, HindIII and KpnI). After hybridization, nine SLA class I genes were identified. By transfection of the four clones into Ltk- cells, the expression of the SLA class I genes was measured cytometrically using an SLA specific antiserum. To differentiate between classical and-non-classical genes, the exons 2 and 3 of the 9 genes were sequenced. After determination of the G/C content at the third codon position and comparison with sequences of published SLA class I genes, three of them could be identified as classical genes. A specific primer was constructed for one of these classical class I genes and used to amplify and differentiate the exon 2 of 200 animals from four different German pig breeds. Zusammenfassung Molekulargenetische Charakterisierung der MHC Klasse I Region beim Schwein Vier unterschiedliche Klone PG2, PG3, PG5 und PG6 wurden durch Screening der Cosmidbank eines in seinen SLA-Haplotypen homozygoten Hausschweines mit einer humanen MHC-Klasse I-cDNA-Sonde isoliert (Hertner 1991). Eine Restriktionskarte fur diese Klone wurde mit vier verschiedenen Endonukleasen (EcoRI, BamHI, HindIII und KpnI) erstellt. Durch Hybridizierungsversuche liesen sich neun SLA-Klasse I-Gene identifizieren. Nach der Transfektion der vier Klone in Ltk- Zellen wurde die Expression der SLA-Klasse I-Gene mit Hilfe eines spezifischen Antiserums cytometrisch gemessen. Um die neun Klasse I-Gene den ‘klassischen’ oder ‘nicht klassischen’ Genen zuzuordnen, wurden die hoch polymorphen Exon 2- und 3-Regionen sequenziert. Nach Bestimmung des G/C-Gehaltes an der dritten Codonposition der sequenzierten Regionen und des Vergleichs mit den publizierten Sequenzen der SLA-Klasse I-Gene wurden drei Gene als ‘klassisch’ zugeordnet. fur ein ‘klassisches’ Klasse I-Gen wurde ein spezifischer Primer konstruiert, mit dem die Exon 2-Region von 200 Tiere aus vier verschiedenen Schweinerassen amplifiziert wurden.

To better characterize the role of the lipoprotein lipase (LPL) gene in the determination of triglyceride levels in healthy subjects, a study was performed in 193 nuclear families (384 parents, means age = 42.0 ± 5.2 years; 399 offspring, mean age = 14.6 ± 4.3 years) volunteering to have a free health checkup examination. The pattern of familial resemblance was compatible with a zero correlation between spouses, a weak father-offspring correlation (0.099 ± 0.054; P < 0.07), and significant mother-offspring (0.235 ± 0.053; P < 10−4) and sib-sib (0.294 ± 0.064; P < 10−4) correlations. Associations of triglyceride levels with the LPL HindIII and PvuII polymorphisms were investigated by a familial measured genotype analysis, specifying sex- and age-dependent polymorphism effects. The effects associated with both polymorphisms were significant only in fathers, the H+ and P+ alleles being associated with raised triglyceride levels. The HindIII and PvuII polymorphisms explained 3.5% and 3%, respectively, of the variability of triglycerides in fathers. The relationship was weakened after prior adjustment on body mass index, but remained significant for PvuII. Because of the lack of effect in mothers and offspring, the polymorphisms did not contribute to the covariance of triglyceride levels in relatives. In conclusion, this family study showed a weak relationship of the HindIII and PvuII polymorphisms to plasma triglyceride levels in young healthy male subjects. The effects detectable only in fathers suggest a possible modulation of the LPL expression by hormonal or lifestyle factors. © 1996 Wiley-Liss, Inc.

Children with malignancy are immunosuppressed and susceptible to serious infections with herpesviruses. The majority of children on chemotherapy for malignancy are seropositive for human herpesvirus-6 (HHV-6), and although HHV-6 has been demonstrated to be a pathogen in severely immunocompromised patients, whether this is the case for paediatric oncology patients is unknown. HHV-6 is secreted in saliva and in this study samples were examined prospectively for HHV-6 DNA in healthy children and those with malignancy. In a nested polymerase chain reaction (PCR), a 287 bp outer fragment and 163 inner fragment of HHV-6 DNA were amplified. The resulting amplimer contained a Hind III restriction site present only in "B" type HHV-6 and this was used to identify the type of HHV-6 amplified. In saliva from healthy control children, 74% (28/38) of samples were HHV-6 DNA-positive in either the supernate, pellet or both. In the patients, 58% (45/77) of all samples were HHV-6 DNA-positive. When sequential samples from twelve patients were examined the children appeared to fall into two groups: those who were frequently HHV-6 DNA-positive (60% of samples or more) and those who were rarely HHV-6 DNA-positive (33% of samples or less) (P < 0.0001). The only apparent difference between these two groups was that the less frequently HHV-6-positive group was more often febrile and unwell with neutropaenia. Hind III digestion demonstrated all the positive samples to be "B" type HHV-6. Possible explanations for this difference in HHV-6 secretion between the patient groups are discussed.

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum-like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using HindIII and EcoRI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917 T , Lact. pentosus ATCC 8041 T , Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei) had identical 16S ribosomal DNA sequences (Lact. plantarum/Lact. pentosus). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensus stricto ; three of these strains had more than 60% DNA : DNA homology to the type strain of Lact. plantarum, and one had less than 50% DNA : DNA homology to Lact. plantarum ATCC 14917 T . Two strains had fragment patterns similar to the type strain of Lact. pentosus, and they had more than 80% DNA : DNA homology to Lact. pentosus ATCC 8041 T . One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of the phenotype and source of isolation. RFLP of the 16S rRNA genes using EcoRI and HindIII might be used for species recognition of Lact. plantarum, but seems less suitable for strain typing.