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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↗

PubMed2003

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism.

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.

Anti-Infective Agents↗

PubMed2005

Limitations on the use of polymerase chain reaction--restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae.

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.

DNA, Fungal↗

PubMed1991

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.

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↗

PubMed2000

Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis.

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism analysis. The method was used to compare ORF 1 sequences of two divergent European-type PRRSV strains. Our results indicated that the structural and replicase parts of these two strains had evolved at overall similar rates.

Blotting, Southern↗

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↗

PubMedDate not supplied

Restriction fragment length polymorphism analysis of highly virulent strains of infectious bursal disease viruses from Holland, Turkey, and Taiwan.

Strains of highly virulent infectious bursal disease virus (HVIBDV) from Turkey (OA, OE), Holland (HOL), and Taiwan (PT, IL) were characterized by reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) analysis and compared with the United States of America (USA) classic (STC) and variant (MD, IN) serotype 1 viruses and serotype 2 OH strain. A primer pair that amplified a 743-bp fragment of the VP2 gene was used. The RFLP profiles of the RT/PCR products were determined with four restriction enzymes (SspI, BstNI, MboI, and StyI). The SspI enzyme distinguished the HVIBDVs from the USA classic and variant viruses and the serotype 2 virus. The USA serotype 1 viruses used in this study did not have restriction sites for SspI in the 743-bp VP2 fragment. The RFLP profiles of the five HVIBDVs when digested with BstNI were different from the USA classic STC virus but were similar to the variant viruses (MD, IN). The RFLP profiles for isolates from Turkey and Holland were similar to the classic STC strain when digested with MboI but were different from the variant USA viruses (MD, IN). The RFLP profiles of the two Taiwan isolates were different from the Holland, Turkey, and USA classic and variant viruses when digested with MboI. When the five HVIBDVs were digested with StyI enzyme, their RFLP patterns were similar to those of the USA classic STC strain but were different from the USA variant viruses. None of the strains tested had patterns similar to serotype 2 OH virus.

Animals↗

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↗

PubMed1992

[DNA polymorphism in the Russian population. Analysis of dna restriction fragment length polymorphism in seven loci of the nuclear genome].

Using the method for polymerase chain reaction the polymorphism of eight markers of the nuclear DNA was studied. In a sample of Russians taken at random (N = 118) from predominantly southern and central regions of Russia, allele frequencies were determined for restriction sites HindIII at HBG-2 and PAH loci, AvaII at the HBB locus, MspI at the ApoB locus, PstI at D7S8, HincII at LDLR, TaqI and BamHI at the DSX164. Comparative data for different world regions are presented.

Chromosome Mapping↗

PubMed2001

Lack of correlation between Mbo I restriction fragment length polymorphism of renin gene and essential hypertension in Japanese.

Predisposition to essential hypertension is associated with gene polymorphisms of the renin angiotensin system (RAS). Gene polymorphisms of the angiotensinogen and angiotensin converting enzyme genes are known to be risk factors for hypertension, while few studies concerning the renin gene polymorphism have been published. In the present investigation, we carried out a case control study using a Japanese population to examine the genetic influence of the renin gene on the predisposition to hypertension. Patients (n=235) recruited from outpatients at Osaka University Hospital and diagnosed with essential hypertension or receiving long-term antihypertensive medication participated in the study. Normotensive control subjects (n=510) without a history of hypertension and without diabetes mellitus were recruited from the same population, and were sex-matched with experimental subjects. A polymorphism in intron 9 of the human renin gene was determined as the Mbo I restriction fragment length polymorphism (Mbo I-RFLP). There was no significant association between Mbo I-RFLP of the renin gene and predisposition to essential hypertension in Japanese (p>0.05, chi2=2.1). These results suggest that the Mbo I (+) allele of the renin gene does not increase the risk for hypertension in Japanese.

Aged↗