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PubMed1988

Carrier detection in severe (type III) von Willebrand disease using two intragenic restriction fragment length polymorphisms.

DNA from a family with a female member affected with severe (type III) vWD was analysed using three restriction enzymes and a partial vWF cDNA probe. Two restriction fragment length polymorphisms (RFLPs) detected with the enzymes Bgl II and Xba I proved to be informative in this family. A 36.0 Kb allele demonstrated with the enzyme Xba I was rare in the general population but very important in this family for segregation analysis of the alleles and their association with the putative defective chromosome. The propositus was homozygous for the 36.0 Kb Xba I polymorphic band and heterozygous for the Bgl II polymorphism. She was the only member of the family showing this allelic pattern. The linkage of the alleles could be determined because her mother was homozygous for the 9.0 Kb Bgl II polymorphism but heterozygous for the Xba I polymorphism. The segregation of the alleles could be traced to the proband's son and a niece. The genotypic analysis revealed that her niece could be considered as carrying a defective gene for severe vWD.

Alleles↗
PubMedDate not supplied

Restriction fragment length polymorphisms at the methylmalonyl CoA mutase locus in normal Chinese.

L-methylmalonyl-CoA mutase (MCM; E.C. 5,4,99,2) is the apoenzyme for catalyzing the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Genetic deficiency of MCM leads to the accumulation of precursors and abnormal metabolites of L-methylmalonyl-CoA. This can be associated with fulminant metabolic acidosis, widespread secondary aberrations in systemic metabolic homeostasis, mental retardation, or even neonatal death. This disorder is termed methylmalonic acidemia (MMA). This report, describes the use of an authentic, full-length cloned human cDNA probe, MCM26, kindly provided by Dr. Fred Ledley, for Southern blot analysis of genomic DNA. The pattern of EcoRI, Sac I and Hind III restriction endonuclease sites is reported from 14 unrelated control individuals of Chinese background. A Southern blot by EcoRI to the MCM26b probe reveals invariant bands of 4.1, 3.8, and 2.2 kb respectively. By EcoRI to the MCM26c probe, 7.2 kb is invariant. By HindIII to the MCM26c probe, invariant bands are 4.8 and 2.7 kb respectively. By SacI to the MCMb probe, invariant bands are 17, 8.0, 6.0, 3.6 and 1.8 kb respectively, while the polymorphic band is at 5.6kb. When combined with more diverse samples and additional polymorphisms, this restriction fragment length polymorphism may be useful for genetic diagnostic and linkage studies of MCM in MMA.

Chromosome Mapping↗
PubMed1992

Analysis of rRNA restriction fragment length polymorphisms from Bacteroides spp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals.

The Escherichia coli rRNA operon rrnB was used as a 32P-labeled hybridization probe in Southern blots of genomic DNAs from representative strains of the saccharolytic, gram-negative, obligate anaerobes of the genus Bacteroides. Control experiments with the B. fragilis type strain ATCC 25285 established that nearly identical rRNA fragment patterns were produced when either the E. coli rrnB gene probe or homologous rRNA isolated from B. fragilis was used as the probe. In addition, it was shown that a specific 16S or 23S rrnB gene probe also could be used to produce fragment patterns suitable for analysis. Thirty-one strains from 8 of the 10 recognized Bacteroides species were then examined. The resulting autoradiographs revealed specific fragment patterns for all but one (B. ovatus) of the species tested. Restriction fragment length polymorphisms were observed for many of the strains tested, but these differences did not hinder species classification. The five B. ovatus strains examined did not form a distinct group, and their rRNA fragment patterns displayed a marked heterogeneity. The same approach was applied to a unique set of enterotoxin-producing B. fragilis strains isolated from animals and humans with diarrhea. The results demonstrated that these strains were in fact B. fragilis and that they produce rRNA fragment patterns closely related to those of the type strain ATCC 25285. This set of strains did not appear to form a separate subgroup or genotype within the B. fragilis species, and there were no distinguishable restriction fragment length polymorphisms that could be used to specifically separate enterotoxin-producing strains from nonenterotoxigenic strains.

Animals↗
PubMed1986

Restriction fragment length polymorphism of the human C3 complement gene.

A cloned gene-specific probe for human complement (C3) was hybridized to DNA samples digested with various restriction endonucleases. The C3 probe detects one restriction fragment length polymorphism (RFLP) that occurs frequently in the French population when DNAs are digested with Sac I. The corresponding DNA alleles can be readily used in linkage analysis of loci on chromosome 19, and such a polymorphism can be followed through myotonic dystrophy families.

Chromosomes, Human, Pair 19↗
PubMedDate not supplied

Restriction fragment length polymorphism (RFLP) heterogeneity of HLA-DQ beta genes associated with DNA fragment identical to the DR1-beta DNA structure.

Restriction fragment length polymorphism (RFLP) analyses of DR1 positive peripheral blood leukocytes DNA was carried out. The Taq I digested DNA was hybridized with cDNA probes for HLA-DR and -DQ beta genes. The DR probe detected fragments commonly observed in the DR1 specificity, whereas a new DQ-beta fragment was detected in some DR1 haplotypes when the DQ-beta probe was used. This fragment had an RFLP pattern identical to the DQ-beta fragment typically associated with most DR2 and some DRw6 specificities.

Alleles↗
PubMed1992

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes.

A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were TaqI and MspI. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.

Animals↗
PubMed1992

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.

Alleles↗
PubMed1992

Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping).

Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.

Adult↗
PubMed1990

Cloning of a DNA fragment displaying restriction fragment length polymorphisms between the genomes of spontaneously hypertensive and Wistar-Kyoto rats.

A 3 kilobase (kb) EcoRI fragment cloned from the genome of the spontaneously hypertensive rat (SHR) displayed restriction fragment length polymorphism (RFLP) compared with the genome of the Wistar-Kyoto rat (WKY) when total genomic Southern blot analysis was performed for two restriction enzymes, PstI and PvuII. Sequencing of the DNA fragment cloned from genomic SHR and WKY libraries revealed that this 3 kb EcoRI fragment harbours three point mutations. Two of them (C to T and A to T) are situated in the middle of the restriction sites for PstI and PvuII, thus disrupting the recognition sites for these enzymes in the SHR genome. Southern blot analysis using total complementary (c) DNA obtained from cDNA libraries of aortic smooth muscle cells from SHR and a whole WKY kidney, with this 3 kb EcoRI fragment as a probe, showed polymorphic bands suggesting that these point mutations are reflected in the sequences of messenger (m) RNA transcribed from the gene encoded in this 3 kb fragment. Detection of two bands by a Northern blot analysis for RNA from various SHR tissues indicates that this 3 kb fragment is actively transcribed in vivo.

Animals↗
PubMed1992

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.

Animals↗
PubMed1989

Definition of DRw10 by restriction fragment length polymorphism.

To better define the presence of the DRw10 haplotype which has sometimes proved difficult to type by using serologic reagents, Southern blot analysis was performed on seven DRw10 heterozygous individuals with rheumatoid arthritis. Using the restriction enzymes Taq I or BamH I, the restriction fragment length polymorphism (RFLP) pattern for the DRw10 haplotype was clearly distinguishable from that of other DR alleles. Digestion with Taq I revealed a unique DR beta/Taq I 12.20 fragment. A characteristic DR beta/Taq I 4.60 fragment was also present only in DRw10 and DR1 haplotypes. Digestion with the restriction enzyme BamH I revealed a DR beta/BamH I 5.07 fragment also present in DRw10 and DR1 haplotypes, and a DR beta/BamH I 4.30 fragment shared with the DRw52 and DR2 haplotypes but not found in DR1 haplotypes. The pattern was readily distinguished from those given by the haplotypes DR4, 7 and w9. Family studies of five individuals demonstrated appropriate segregation of the restriction fragments. In particular, segregation of DRw10 haplotypes from DR1 haplotypes was clearly shown in a family in which the DRw10 haplotype was associated with rheumatoid arthritis in two individuals. Southern blot analysis proved to be a useful alternative method for identifying the DRw10 allele in certain combinations where this allele has been difficult to define serologically.

Arthritis, Rheumatoid↗
PubMed1989

HLA-D typing of heterozygotes using restriction fragment length polymorphism in the HLA-DQ gene region on the basis of standard band patterns derived from HLA homozygotes.

Genomic DNAs from 37 Epstein-Barr virus-transformed HLA-D homozygous cell lines with different Dw specificities, Dw1-Dw23, and DKT2 were digested with four different restriction endonucleases (EcoRI, PstI, TaqI, and MspI) and hybridized to DQ alpha and DQ beta cDNA probes. Polymorphic patterns of multiple fragments correlating with Dw, DQ, and DR haplotypes were detected, and the restriction fragment length polymorphism standard pattern specific for each Dw, DQ, and DR specificity could be defined. The polymorphic fragment patterns of HLA-D heterozygotes were predicted simply by the summation of two standard patterns of HLA-D homozygotes and utilized to identify HLA-D specificities of 25 normal individuals, who are HLA heterozygotes in most cases. The HLA-D, -DR, and -DQ specificities defined by this DNA typing were compared with those assigned by serologic and cellular typing. There was good correlation, allowing the application of accurate genotyping by DQ alpha and DQ beta cDNA probes to HLA class II typing of HLA heterozygotes.

Base Sequence↗
PubMed1989

DQA1 restriction fragment length polymorphisms and insulin-dependent diabetes mellitus: a BglII fragment labels a subset of B8,DR3 haplotypes uniquely associated with insulin-dependent diabetes mellitus.

Class II restriction fragment length polymorphism studies of 38 pedigrees with multiple cases of insulin-dependent diabetes mellitus revealed the existence of a DQA1-related polymorphism that distinguishes two kinds of HLA-B8,DR3 haplotypes. One of these, characterized by the presence of DQA1-BglII 7.20 kb, was present in all 14 examples inherited by patients and in 6 of the 12 B8,DR3 haplotypes not so inherited. The apparently complete association between the presence of this fragment and the "affected" status of B8,DR3 haplotypes (p = 0.004) was confirmed in a separate group of 26 simplex pedigrees selected for the presence of this haplotype in the respective probands (combined p less than 0.0001).

Diabetes Mellitus, Type 1↗
PubMed1988

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha- and beta-chain cDNA probes.

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.

Animals↗
PubMed1990

Growth hormone restriction fragment length polymorphisms that segregate with 42-day live weight of mice.

The known correlation between growth hormone levels and growth rate in a number of species prompted us to examine if polymorphic restriction fragment alleles at the growth hormone locus in mice might be associated with differentiable rates of growth. An F2 population of mice was generated from crosses between a line selected for high 42-day weight and an unselected control line. The original selected and control lines exhibited mean 42-day weights of 30.6 +/- 3.8 and 20.5 +/- 2.6 g, respectively. Since the two lines also differed with respect to the restriction fragments detected by hybridization to a rat growth hormone cDNA probe, an analysis of the F2 generation was carried out to determine whether this polymorphism could be considered a quantitative trait locus for 42-day weight. The results of the analysis indicated that a polymorphic HindIII restriction fragment was correlated (P less than 0.05) with 42-day weight. However, the allele that was positively correlated with weight was the one that was fixed in the original control line, rather than the one from the selected line. While these findings support the potential use of restriction fragment length polymorphisms in quantitative trait evaluation of livestock, they also emphasize the requirement for testing such potential quantitative trait loci in the appropriate genetic background.

Animals↗
PubMed1989

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines.

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.

Animals↗