scholarly journals Toxin-deficient mutants from a toxin-sensitive transformant of Cochliobolus heterostrophus.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 751-757
Author(s):  
G Yang ◽  
B G Turgeon ◽  
O C Yoder
Keyword(s):  
Fragment Length Polymorphism ◽  
Signal Sequence ◽  
Single Gene ◽  
Toxin Production ◽  
Cochliobolus Heterostrophus ◽  
Polygalacturonic Acid ◽  
Wild Type ◽  
Transformation Vector ◽  
Screening Procedures ◽  
Restriction Fragment Length

Abstract Tox1 is the only genetic element identified which controls production of T-toxin, a linear polyketide involved in the virulence of Cochliobolus heterostrophus to its host plant, corn. Previous attempts to induce toxin-deficient (Tox-) mutants, using conventional mutagenesis and screening procedures, have been unsuccessful. As a strategy to enrich for Tox- mutants, we constructed a Tox1+ strain that carried the corn T-urf13 gene (which confers T-toxin sensitivity) fused to a fungal mitochondrial signal sequence; the fusion was under control of the inducible Aspergillus nidulans pelA promoter which, in both A. nidulans and C. heterostrophus, is repressed by glucose and induced by polygalacturonic acid (PGA). We expected that a transformant carrying this construction would be sensitive to its own toxin when the T-urf13 gene was expressed. Indeed, the strain grew normally on medium containing glucose but was inhibited on medium containing PGA. Conidia of this strain were treated with ethylmethanesulfonate and plated on PGA medium. Among 362 survivors, 9 were defective in T-toxin production. Authenticity of each mutant was established by the presence of the transformation vector, proper mating type, and a restriction fragment length polymorphism tightly linked to the Tox1+ locus. Progeny of each mutant crossed to a Tox1+ tester segregated 1:1 (for wild type toxin production vs. no or reduced toxin production), indicating a single gene mutation in each case. Progeny of each mutant crossed to a Tox1- tester segregated 1:1 (for no toxin production vs. no or reduced toxin production) indicating that each mutation mapped at the Tox1 locus. Availability of Tox- mutants will permit mapping in the Tox1 region without interference from a known Tox1 linked translocation breakpoint.

scholarly journals BSMI AND TAQI POLYMORPHISMS IN VITAMIN D RECEPTOR GENE OF TYPE 2 DIABETES MELLITUS PATIENTS FROM NORTH INDIA

2016 ◽  
Vol 9 (9) ◽  
pp. 186 ◽  
Author(s):  
Nancy Taneja ◽  
Rajesh Khadagawat ◽  
Shalini Mani
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Vitamin D ◽  
Vitamin D Receptor ◽  
Metabolic Diseases ◽  
Fragment Length Polymorphism ◽  
Wild Type ◽  
Significant Difference ◽  
Restriction Fragment Length

ABSTRACTObjective: Polymorphisms in vitamin D receptor (VDR) genes are known to be linked with different metabolic diseases including Type 2 diabetesmellitus (T2DM) also. However, the association of these polymorphisms is not much explored for the Indian population. To determine the prevalenceof BsmI and TaqI polymorphism in VDR gene of T2DM patients from North India.Methods: Blood samples were obtained from 100 well-characterized T2DM patients and 100 healthy controls. Genomic DNA was isolated from bloodsamples and using polymerase chain reaction/restriction fragment length polymorphism based method, the presence of these polymorphisms wasinvestigated in these samples. The data were statistically analyzed using SPSS 21.0 software.Results: For TaqI polymorphism, both the wild type (TT) and heterozygous (TC) genotype showed a significant difference between patients andcontrols (p=0.023 and p<0.001, respectively). Whereas, the frequency of CC genotype was not significantly different among these groups (p=0.506).For BsmI polymorphism also, the frequency of wild type (GG) and heterozygous (GA) genotype was significantly different in patients and controls(p=0.027 and p=0.001), respectively. However, the frequency of AA genotype was not of statistical significance in patients (p=0.071).Conclusions: The mutant alleles of TaqI and BsmI polymorphisms are known to be associated with different metabolic diseases, including diabetestoo. In our study also, there is a significant difference between the frequency of wild type and heterozygous genotype for these polymorphisms. Thissuggests that BsmI and TaqI polymorphisms may be associated with T2DM patients.Keywords: Type 2 diabetes mellitus, Polymorphism, Vitamin D receptor, Patient, Control, Restriction fragment length polymorphism.

scholarly journals Assessment of Poliovirus Eradication in Japan: Genomic Analysis of Polioviruses Isolated from River Water and Sewage in Toyama Prefecture

2000 ◽  
Vol 66 (11) ◽  
pp. 5087-5091 ◽  
Author(s):  
Kumiko Matsuura ◽  
Mitsuhiro Ishikura ◽  
Hiromu Yoshida ◽  
Takashi Nakayama ◽  
Sumiyo Hasegawa ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
River Water ◽  
Viral Genome ◽  
Fragment Length Polymorphism ◽  
Genomic Analysis ◽  
Rflp Analysis ◽  
Wild Type ◽  
Pcr Rflp ◽  
Nucleotide Divergence ◽  
Restriction Fragment Length

ABSTRACT Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.

MUTATION IN THE FMO3 LOCUS AND ITS DISTRIBUTION IN A GROUP OF AYRSHIRE BREEDING BULLS USED IN FARMS OF THE KRASNODAR TERRITORY

2021 ◽  
pp. 10-15
Author(s):  
Н.В. КОВАЛЮК ◽  
Е.В. ШИРЯЕВА ◽  
Л.И. ЯКУШЕВА ◽  
Ю.Ю. ШАХНАЗАРОВА
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutant Allele ◽  
Restriction Site ◽  
Fragment Length Polymorphism ◽  
Test System ◽  
Frequency Of Occurrence ◽  
Wild Type ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Рыбный привкус в коровьем молоке вызван наличием нонсенс-мутации (g.39523051C>T) в гене бычьего FMO3. Нами разработана тест-система для выявления FMO3- полиморфизма, основанная на полимеразной цепной реакции с последующим анализом полиморфизма длин фрагментов рестрикции с использованием эндонуклеазы TaqI. Фрагменты, которые амплифицировались с участка гена FMO3 «дикого» типа, расщеплялись эндонуклеазой TaqI  на 2 фрагмента: 136 и 99 пн. Фрагменты, амплифицированные с мутантного аллеля, сайта рестрикции не имели (их размер составлял  235 пн). Определена частота встречаемости носителей мутации в отечественной субпопуляции айрширского скота. Установлено, что среди айрширских быков-производителей (n=45), принадлежащих различным отечественным и зарубежным племорганизациям, частота встречаемости носителей мутации в гене FMO составила 9%. Учитывая, что выявленные носители мутации интенсивно используются и могут передавать эту аномалию значительному числу дочерей, генотипирование по локусу FMO3 должно стать обязательным для быков-производителей и групп быкопроизводящих коров племенных айрширских хозяйств. The fishy taste in cow's milk is caused by the presence of a nonsense mutation (g.39523051C>T) in the bovine FMO3 gene. We have developed a test system for detecting FMO3 polymorphism based on a polymerase chain reaction followed by analysis of restriction fragment length polymorphism using TaqI endonuclease. Fragments that were amplified from the wild-type FMO3 gene site were cleaved by TaqI endonuclease into 2 fragments: 136 and 99 bp. The fragments amplified from the mutant allele did not have a restriction site (their size was — 235 bp). The frequency of occurrence of mutation carriers in the domestic subpopulation of Ayrshire cattle was determined. It was found that among Ayrshire bulls (n=45) belonging to various domestic and foreign breeding organizations, the frequency of occurrence of carriers of the mutation in the FMO gene was 9%. Given that the identified carriers of the mutation are intensively used and can transmit this anomaly to a significant number of daughters, genotyping by the FMO3 locus should become mandatory for breeding bulls and groups of bull-producing cows of breeding Ayrshire farms.

scholarly journals A restriction fragment length polymorphism map and electrophoretic karyotype of the fungal maize pathogen Cochliobolus heterostrophus.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 81-96 ◽  
Author(s):  
T H Tzeng ◽  
L K Lyngholm ◽  
C F Ford ◽  
C R Bronson
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Genome Size ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Cochliobolus Heterostrophus ◽  
Electrophoretic Karyotype ◽  
Rflp Map ◽  
Restriction Fragment Length

Abstract A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.

Association of the NAD(P)H-bispecific nitrate reductase structural gene with the Nar7 locus in barley

Genome ◽  
1995 ◽  
Vol 38 (4) ◽  
pp. 743-746 ◽  
Author(s):  
R. L. Warner ◽  
D. A. Kudrna ◽  
A. Kleinhofs
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Nitrate Reductase ◽  
Structural Gene ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Reductase Activity ◽  
Wild Type ◽  
Restriction Fragment Length

The NADH-specific and NAD(P)H-bispecific nitrate reductase genes from barley have been cloned and sequenced. To determine if the Nar7 locus encodes the NAD(P)H-bispecific nitrate reductase structural gene, a cross was made between a wild-type cultivar, Morex (Nar7 Nar7), and Az70 (nar7w nar7w), a mutant from the cultivar Steptoe that is deficient in NAD(P)H-bispecific nitrate reductase activity. A probe specific to the NAD(P)H-bispecific nitrate reductase structural gene detected restriction fragment length polymorphism between the parents. This probe was used to classify selected F2 progeny for restriction fragment length genotype. All the NAD(P)H nitrate reductase deficient F2 progeny (24/101) possessed the Az70 restriction fragment genotype. The absence of recombination between the NAD(P)H-bispecific nitrate reductase deficient genotype and the NAD(P)H-bispecific nitrate reductase restriction fragment length genotype indicates that the two traits are closely associated in inheritance and that Nar7 is probably the NAD(P)H-bispecific nitrate reductase structural gene.Key words: Hordeum vulgare, nitrate reductase, restriction fragment length polymorphism.

scholarly journals Deletion of all Cochliobolus heterostrophus Monofunctional Catalase-Encoding Genes Reveals a Role for One in Sensitivity to Oxidative Stress but None with a Role in Virulence

2003 ◽  
Vol 16 (11) ◽  
pp. 1013-1021 ◽  
Author(s):  
Barbara Robbertse ◽  
O. C. Yoder ◽  
Anita Nguyen ◽  
Conrad L. Schoch ◽  
B. Gillian Turgeon
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Signal Sequence ◽  
Large Subunit ◽  
Small Subunit ◽  
Cochliobolus Heterostrophus ◽  
Wild Type ◽  
Enhanced Sensitivity ◽  
Encoding Genes ◽  
Harmful Effects

The genome of the maize pathogen Cochliobolus heterostrophus encodes three unlinked monofunctional catalase-encoding (CAT) genes that singly or in combination could offer protection against the harmful effects of oxidative stress. Phylogenetic analysis placed the CAT2 and CAT3 proteins in a cluster with large subunit catalases (CAT3 has a secretory signal sequence and was grouped with known secreted catalases), whereas CAT1 clustered with small subunit catalases. Single, double, and triple cat mutants were created and screened for sensitivity to hydrogen peroxide and altered virulence on maize. All mutants deficient in CAT3 had enhanced sensitivity to hydrogen peroxide, as compared with wild type or with mutants deficient in CAT1, CAT2, or both. All catalase-deficient mutants had normal virulence to maize. Thus, the secreted CAT3 protein protects the fungus from oxidative stress during vegetative growth, but members of this enzyme family, alone or in combination, are not essential for virulence.

scholarly journals Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation

2021 ◽  
Author(s):  
Sirwan M.A. Al-Jaf ◽  
Sherko Subhan Niranji ◽  
Zana Hameed Mahmood
Keyword(s):  
Fragment Length Polymorphism ◽  
Taqman Probe ◽  
Nasopharyngeal Swab ◽  
Amplification Refractory Mutation System ◽  
Common Mutation ◽  
Wild Type ◽  
Chain Reaction ◽  
Mutation System ◽  
Polymerase Chain ◽  
Restriction Fragment Length

A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in impacting of this mutation on the virus's infectivity, and the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutant indicated by presence of HEX probe and 176bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of G614 mutant. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.

Ki-ras mutations as a prognostic factor in extrahepatic bile system cancer. PANK-ras I Project Investigators.

1995 ◽  
Vol 13 (7) ◽  
pp. 1679-1686 ◽  
Author(s):  
N Malats ◽  
M Porta ◽  
J L Piñol ◽  
J M Corominas ◽  
J Rifà ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Fragment Length Polymorphism ◽  
Prognostic Significance ◽  
Prognostic Indicator ◽  
Survival Duration ◽  
Tumor Site ◽  
Wild Type ◽  
Ras Mutations ◽  
Artificial Restriction ◽  
Restriction Fragment Length

PURPOSE To assess the prevalence and prognostic significance of Ki-ras codon 12 mutations in extrahepatic biliary system cancer (EBSC). PATIENTS AND METHODS Patients diagnosed with EBSC between 1980 and 1990 (N = 111) were selected from two hospitals. DNA was amplified from paraffin-embedded tissues and mutations in codon 12 of Ki-ras were detected using the artificial restriction fragment-length polymorphism (RFLP) technique. RESULTS Tissue was available from 68.5% of patients. The prevalence of mutations was 41%. There was no association between mutations and clinical and pathologic characteristics; however, mutations in Ki-ras were associated with survival, with a median survival duration of 7.7 months for patients with wild-type Ki-ras and 1.7 months for patients with mutated tumors (hazards ratio [HR] = 1.67; P = .075). Among patients with stage I to II tumors, the chance of dying of patients with the mutation was 7.8 times higher than that of patients without the mutation (P = .087); the corresponding HR for patients with stage III to IV disease was 2.9 (P = .003). After adjusting for age, tumor site, histology, differentiation, and stage, the HR for Ki-ras mutations was 2.12 (P = .026). CONCLUSION Ki-ras codon 12 mutations are an independent prognostic indicator in patients with EBSC. Mutation detection may be of help in the management of these patients.

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