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PubMed1991

Distinction of deep versus superficial clinical and nonclinical isolates of Trichosporon beigelii by isoenzymes and restriction fragment length polymorphisms of rDNA generated by polymerase chain reaction.

Fifteen clinical and environmental strains of Trichosporon beigelii were analyzed for similarities by using morphological features, biochemical profiles based on carbon compound assimilation and uric acid utilization, isoenzyme electrophoresis, and restriction fragment length polymorphisms of a segment of genes coding for rRNA expanded with the polymerase chain reaction. The findings suggest that strains that cause invasive disease are distinct from the superficial and the nonclinical isolates and that isolates from the skin and mucosae represent a number of different organisms, including some environmental forms. The study shows that T. beigelii is a complex of genetically distinct organisms and that more than one type is found in clinical samples.

Base Sequence↗

PubMed1986

Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4.

Chromosome Banding↗

PubMed2004

[Relations between clinical subtypes of Mycobacterium avium pulmonary disease and polyclonal infections detected by IS1245 based restriction fragment length polymorphism analysis].

INTRODUCTION: The epidemiology of Mycobacterium avium-intracellulare (MAC) infections has not been completely defined. Recently some reports presented polyclonal MAC infections. The purpose of this study was to reveal the clonal diversity of Mycobacterium avium isolates and the relation between clinical subtype of lung disease and polyclonal infection. METHODS: We categorized pulmonary Mycobacterium avium infection to three clinical subtypes, tuberculosis like type, bronchiectasis with preexisting tuberculosis type and nodular bronchiectasis type. Mycobacterium avium isolates of 11 patients were studied for their heterogeneity using IS1245 based RFLP analysis. The insertion sequence IS1245 is repetitive element identified only in Mycobacterium avium. Standard method of IS1245 based RFLP analysis has been proposed as a suitable technique for typing of Mycobacterium avium isolates for epidemiological and taxonomic studies. At least three distinct colonies were subcultured to single clone. The subclones of the isolates were analyzed by IS1245 based RFLP technique and some subclones were also examined by antimicrobial susceptibility test. RESULTS: Two of three patients of tuberculosis like type were considered to be monoclonal infection because only a single genotype was identified. And only one of four patients of bronchiectasis with preexisting tuberculosis type was considered to be polyclonal infection despite of long-term observation. Although isolates were collected in two or more occasions in clinical course over one year period, only a single genotype was observed in two patients. In contrast, three of four patients of nodular bronchiectasis type had multiple genotypes. Isolates recovered from patients with monoclonal infection pattern following long-term treatment with clarithromycin monotherapy became resistant to clarithromycin. In contrast, three strains derived from one nodular bronchiectasis patient were susceptible to clarithromycin despite of long-term chemotherapy including clarithromycin. The susceptibility patterns of the other drugs were also apparently different. Strain conversion due to repeated polyclonal infection was considered. These results of the antimicrobial susceptibility test supported clonal diversity of the Mycobacterium avium infection. DISCUSSION: IS1245 based RFLP analysis possesses a discriminatory power between the isolates on clonal level. This study demonstrates that polyclonal infections are common in nodular bronchiectasis type and monoclonal infections are common in tuberculosis like type and bronchiectasis with preexisting tuberculosis type. And not only simultaneous polyclonal infection but also repeated polyclonal infection were observed in a nodular bronchiectasis type patient. Drug susceptibility test showed long-term chemotherapy including clarithromycin could change the susceptibility of clarithromycin to resistant in patients with monoclonal infection. In contrast patients with repeated polyclonal infection pattern would avoid drug resistance because of strain conversion. This multiple susceptibility patterns identified in this study would not have been detected by the standard susceptibility test without subculture. And we also need the treatment strategy considering the polyclonal infection. CONCLUSIONS: Polyclonal infections are considered to be common in pulmonary Mycobacterium avium infection, especially nodular bronchiectasis type. Clonal diversity of Mycobacterium avium infection is an important factor to perform chemotherapy and drug susceptibility test.

Adolescent↗

PubMed2002

PCR restriction fragment length polymorphism in detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine.

OBJECTIVE: To analyse the emergence of YMDD motif (tyrosine-methionine-aspartate-aspartate) variants in patients with hepatitis B treated with lamivudine. METHODS: The amino acid substitution from methionine or isoleucine at the YMDD motif at the HBV polymerase gene is a main mutation resistant to lamivudine treatment. Generated from a fragment of domain C of the polymerase gene, patients' HBV DNA, which had been positive previously became positive again ever since it had been negative during lamivudine therapy. Variants were detected by cleavage of the products of the three PCRs with following enzymes: FokI, SspI, Alw441. The results of PCR-RELP were analysed by 8.4% polypropylene acidemide gel electrophoresis. PCR-RFLP assay was compared to direct sequencing. RESULTS: HBV DNA was positive again in 33 patients and positive for one year in 2 patients. YMDD variants were detected in serum 14 of 35 patients, YIDD variants in 4, YVDD variants in 6, and YI/MDD variants in 1; all were in concordance with the results of direct sequencing. The samples of other 3 patients showed YI/VDD mutations, as shown by direct sequencing. The results of PCR-RFLP assay of the mixed sera of YIDD and YVDD variants were similar to those sera of YI/VDD variants. CONCLUSION: PCR-RFLP is suitable for rapid detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine.

Adolescent↗

PubMed1994

TaqI restriction fragment length polymorphisms for growth hormone in bovine breeds and their association with quantitative traits.

The objectives of the present study were (1) to reveal GH-TaqI RFLPs in different bovine breeds and (2) to look for association between quantitative traits and GH-TaqI RFLPs in bulls highly selected for growth and meat production. Blood was sampled from 269 Holstein heifers, 48 Italian and 45 Hungarian Simmental heifers, 30 Normande heifers and 41 bulls and 13 heifers of the double-muscled Belgian White Blue breed. Bulls were weighed from 7 until 13 months of age and the average daily gain, the feed efficiency, the height and the phenotypic index were determined at the end of the experiment. GH-TaqI RFLPs revealed 4 DNA fragments of 6 (A), 5.2 (B), 4.4 (C) and 4.2 (D) kbp. The distribution of genotypes was different between Normande and the other breeds (P < 0.001) and between Belgian White Blue and Holstein (P < 0.001) or Hungarian Simmental breed (P < 0.005). The Normande heifers were also characterized by a high frequency of the AB genotype (53%) compared with other experimental breeds (range values: 7-24%). In the statistical analysis of the relationships between quantitative traits and GH-TaqI RFLPs in double-muscled Belgian White Blue bulls, the AC genotypic class was not used because of its low distribution. The AA class showed greater records of weight at 7 (P < 0.07) and 13 (P < 0.06) months of age than the AB class. In conclusion, this study has shown a correlation between the GH-TaqI RFLPs and weight at 7 and 13 months of age in Belgian White Blue bulls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Analysis of MHC class II and class IV restriction fragment length polymorphism in chicken lines divergently selected for multitrait immune response.

In the present study, chickens of four lines divergently selected for high (H) and low (L) immunocompetence in replicate were analyzed to investigate polymorphisms of MHC class II and MHC class IV on the molecular level associated with selection. The long-term selection experiment for multitrait immunocompetence was carried out in replicates and allows, therefore, the opportunity to distinguish effects of selection from other genetic factors. The SacI-digested DNA was hybridized individually with MHC class II and MHC class IV gene probes. The MHC class II RFLP analysis revealed four polymorphic bands and only one of them showed a significant difference between the selection directions H and L pooled between replicates. The small frequency differences of this band relative to the long-term selection suggest that this MHC class II fragment may contain genetic elements that are only slightly associated with the immune response traits used for selection. The hybridization with the MHC class IV probe displayed 26 scorable bands, of which 18 were polymorphic. In most instances, the differences between the lines were likely caused by the influence of genetic factors other than selection for multitrait immunocompetence. Only one band displayed a consistency in difference between selection directions in both replicates and no frequency difference between replicates. This band was almost completely absent in both H sublines, but at a frequency of about 50% in both L sublines. The general results of this study did not reveal major differences in band frequencies that indicate a close association of MHC class II and MHC class IV polymorphic markers to the divergent selection for multitrait immune response. Although the MHC makes a crucial contribution in immune response, it may have been difficult to detect single-gene associations with the selection criteria of this study, because of the myriad of components contributing to general immune responses measured in vivo.

Animals↗

PubMed1988

Restriction fragment length polymorphisms detected by anonymous DNA probes mapped to defined intervals of human chromosome 16.

Three anonymous DNA probes ACH207, ACH224, and ACH202, isolated from a flow-purified chromosome 16 library and mapped to defined intervals of human chromosome 16, detected restriction fragment length polymorphisms (RFLPs). The RFLPs were of simple two allele types. The ACH207 (D16S4) probe detected a TaqI and an MspI RFLP with polymorphism information content (PIC) values of 0.30 and 0.27; the ACH224 (D16S5) probe detected an RsaI RFLP, PIC value of 0.34; and the ACH202 (D16S4) probe detected an XbaI RFLP, PIC value of 0.22.

Alleles↗

PubMed2004

[Molecular identification methods of yeasts of biotechnological interest].

Yeasts have numerous applications in modern and traditional biotechnology. They take place in production of food, unicellular protein and products with added value, and in the last decades they have been incorporated to the biotechnology industry as host in the production of eukaryotes proteins. Apart from their advantages, some genera are the causes of mycosis on man and in some cases, are opportunistic pathogens associated to diseases such as HIV. They are also agents responsible for the damaging of fresh and elaborated food for human consumption. For these reasons, the quick and accurate identification of industrially, environmentally and clinically significant yeasts is important. Yeast taxonomy has been supported by conventional techniques, based on morphological and physiological descriptions of species and genera, but depend on strain culture conditions, therefore they have introduced errors in yeast taxonomy and originated the duality of their nomenclature. These difficulties have been solved with the application of molecular techniques, based on the sequence analysis of nucleic acid, specially karyotiping electrophoresis, microsatellite analysis, mitochondrial DNA length polymorphism, restriction fragment length polymorphism of ribosomal RNA, ramdom amplified polymorphic DNA and low molecular weight RNA. In this review all those methods are described, which have allowed the development of identification kits for clinical and industrial application for the clearance of phylogenetic relationships among species and genera of yeasts of biotechnological interest.

Biotechnology↗

PubMed1991

Schistosoma mansoni: use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata.

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely Bg/II, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.

Animals↗

PubMed1996

Allelic variants of human TCR BV17S1 defined by restriction fragment length polymorphism, single strand conformation polymorphism, and amplification refractory mutation system analyses.

Several human TCR BV gene subfamilies, including BV3, BV14, and BV17S1, are single member genes but are overutilized among activated CD4+ synovial T cells in the rheumatoid arthritis (RA). To define the role of these TCR BV genes in the pathogenesis of disease, it is critical to characterize the genomic organization and the allelic variations of these genes. In this study we describe allelic variations of BV17S1 defined by restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), and amplification refractory mutation system (ARMS) analyses. A single nucleotide replacement (C/T) results in an amino acid substitution (F/L) in the leader and distinguishes BV17S1*1 from BV17S1*2. This nucleotide substitution was found to create a BsmAI restriction enzyme recognition site in BV17S1*2. Therefore genotypic analyses can be performed either by the SSCP or RFLP method. The analyses of 75 unrelated individuals show that the frequency for allele BV17S1*1 is 52.7% and for allele BV17S1*2 is 47.3%. Both alleles are functionally expressed and are distributed within CD4+/CD8+ T cell subsets. Another point mutation in the CDR2 region of BV17S1, which results in the amino acid replacement of Gln by His, originally identified form a cDNA clone, has now been confirmed as an allele by ARMS analysis using genomic DNA preparations and designated to as BV17S1*3. Screening of this CDR2 related variant among normal populations indicates that this is a rare allele (1 of 75). Although this variant may be of functional significance, the genotypic analysis and functional studies are difficult due to the low frequency of BV17S1*3. In an attempt to define a correlation between BV17S1 allelic usage and susceptibility to RA, the germline distribution of BV17S1 alleles *1 and *2 has been examined in a small number of RA patients and no skewed usage has been identified.

Alleles↗

PubMed2005

Detection of cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism.

Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.

Animals↗

PubMed1991

Detection of frequent BglII polymorphism by polymerase chain reaction and TaqI restriction fragment length polymorphism for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase at the human HSD beta 3 locus (1p11-p13).

Analysis of amplified polymerase chain reaction products of 575 bp from the fourth exon of the human type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase gene at locus HSD beta 3 1p11-p13, reveals a frequent two-allele polymorphism at codon Leu338 due to a silent substitution of T by C, thus creating a BglII site leading to 371- and 204-bp fragments. Southern blot analysis of BglII-digested DNA from 57 individuals using a genomic probe detects two allelic fragments of 5.3 kb and 0.77 kb, respectively, while two allelic fragments of 3.7 kb and 3.4 kb are obtained in TaqI digests with multiple constant bands, as also observed with BglII digests.

Bacterial Proteins↗

PubMed2002

Close correlation between restriction fragment length polymorphism of L-myc gene and susceptibility to gastric cancer.

AIM: A common inherited RFLP of the L-myc proto-oncogene has been reported to correlate with cancer susceptibility. Our aim was to test the hypothesis that there was association between L-myc S allele in gastric cancer and predisposition to the disease. METHODS: The distribution of L-myc polymorphism in 25 patients with gastric cancer was determined by polymerase chain reaction-based restriction fragment length polymorphism and compared with that of 83 healthy control subjects. RESULTS: We found a significant difference, both in the distribution of the LL, LS and SS genotypes and in the allelic frequencies, between the control group and the patient group; that is, the frequencies of L-myc alleles were, L and S, 0.52 and 0.48, 0.64 and 0.36, respectively. This difference was primarily the result of a high frequency of the S allele among gastric cancer patients compared to controls. There was a significant difference in the distribution of both genotypes (P = 0.004) and allele frequencies (P = 0.005) between patients with gastric cancer and control groups. CONCLUSIONS: Our results suggested that L-myc polymorphism may be significant in an individual's susceptibility to gastric cancer in Turkey and may be useful for identifying patients at high risk of developing gastric cancer.

Alleles↗

PubMed1998

Association between restriction fragment length polymorphism of the L-myc gene and susceptibility to gastric cancer.

BACKGROUND: L-myc polymorphism has been documented to be a representative genetic trait which is related an individual's susceptibility to several cancers. However, there have been no reports concerning any significant association between susceptibility to gastric cancer and L-myc polymorphism. METHODS: The distribution of L-myc polymorphism in 61 patients with gastric cancer was determined by polymerase chain reaction-based restriction fragment length polymorphism and compared with that of 107 healthy control subjects. RESULTS: There was a significant difference in the distribution of both genotypes (P = 0.024) and allele frequencies (P = 0.026) between the two groups. The relative risk of gastric cancer for genotypes with the shorter (S) allele was 3.09 compared with the longer (L) allele homozygote. No significant correlation with clinicopathological features of the cancers except for prognosis was found. The patients with SS genotypes had a worse prognosis than those with LL or LS genotypes (P = 0.029). CONCLUSION: L-myc polymorphism may be significant in an individual's susceptibility to gastric cancer in Japan, and may be a useful marker for identifying patients at high risk of developing gastric cancer.

Disease Susceptibility↗

PubMed2001

IGHV3-associated restriction fragment length polymorphisms confer susceptibility to bullous pemphigoid.

OBJECTIVE: To determine whether the immunoglobulin heavy chain genes contribute to the occurrence of bullous pemphigoid (BP), polymorphisms of both the immunoglobulin constant IGHC and variable IGHV groups were studied in 100 Caucasian BP patients and 143 ethnically matched healthy individuals. METHODS: To analyze the restriction fragment length polymorphism (RFLP) of the IGHG constant locus, a genomic immunoglobulin gamma 3 probe which detects polymorphisms in the gamma constant genes was hybridized to BstEII- or BamHI/SacI-digested germline DNA, while IGHV3 subgroup polymorphism was analyzed by hybridizing a cloned VH3 probe to EcoRI-digested DNA. RESULTS: No difference in the frequencies of the genotypes defined by the constant probe was observed between patients and controls. Analysis of the RFLP obtained with the VH3 probe showed that within the range of 4.5 and 1.5 kb, the observed band pattern was composed of 8 monomorphic and 7 polymorphic bands. Among the latter, 4 allowed to define 10 different restriction patterns. One pattern was shown to be significantly less frequent in patients than in controls. CONCLUSION: IGHV3 polymorphism might be a factor conferring susceptibility to BP.

Deoxyribonucleases, Type II Site-Specific↗

PubMed1991

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.

Arylamine N-Acetyltransferase↗

PubMed2000

L-myc restriction fragment length polymorphism in Japanese patients with esophageal cancer.

L-myc polymorphism is a representative genetic trait related to an individual's susceptibility to several cancers. However, there have been no reports concerning the association between esophageal cancer and L-myc polymorphism. To analyze the distribution of polymorphism in Japanese patients with esophageal cancer, a molecular genotyping method using a polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) was used. Based on an analysis of 65 Japanese patients with esophageal cancer and 107 healthy control subjects, a significant difference was observed in either the distribution of genotypes (P=0.012) or of allele frequencies between the two groups (P=0.004). The relative risk of esophageal cancer for genotypes including the shorter allele was 2.9 compared to the longer allele homozygote. Furthermore, the patients with S-allele had a tendency for poor prognosis among those with three genotypes. A significant difference between the distribution of genotypes and the incidence of lymph node metastasis was found based on the clinicopathological features of the cancers. These results suggest that L-myc polymorphism may be implicated as a genetic trait affecting an individual's susceptibility to esophageal cancer, at least among Japanese patients.

Esophageal Neoplasms↗

PubMed1999

Advantages of dental mitochondrial DNA for detection and classification of the sequence variation using restriction fragment length polymorphisms.

After amplification by polymerase chain reaction (PCR), the nucleotide sequences of a 452-bp section of the D-loop region of human mitochondrial DNA (mtDNA) were determined in 40 teeth extracted from patients living in Kanagawa prefecture, Japan. Dental DNA was extracted separately from the dental pulp and dentin (i.e., the attached pulp cells from the most superficial layer of the pulp cavity wall) of the same tooth. Comparison of the nucleotide sequences of the 452-bp region of the D-loop demonstrated that nucleotide substitutions and insertion/deletion events were identical in material from both sources. Thus, dentin produces equivalent results when the dental pulp of a tooth is unsuitable for mtDNA analysis. To establish the reliability of the screening procedure for the sequence analysis, we identified restriction sites for the enzymes KpnI and MnlI in the 452-bp region of the D-loop. Thirteen of 14 patterns of four polymorphisms analyzed using the mtDNA from the 40 tooth samples were identifiable by an initial screening procedure involving restriction fragment length polymorphism (RFLP) analysis. Combined use of sequence analysis and RFLP analysis proved extremely efficient in analyzing mtDNA polymorphisms, allowing identification of individuals.

Adolescent↗