scholarly journals Identification of β-Glucosidase Aggregating Factor (BGAF) and Mapping of BGAF Binding Regions on Maize β-Glucosidase

2000 ◽  
Vol 276 (15) ◽  
pp. 11895-11901 ◽  
Author(s):  
David J. Blanchard ◽  
Muzaffer Cicek ◽  
Jialun Chen ◽  
Asim Esen
Keyword(s):  
Null Allele ◽  
Terminal Region ◽  
Binding Assays ◽  
Terminal Sequence ◽  
Chain Reaction ◽  
Chimeric Enzymes ◽  
Repeat Units ◽  
Maize Genotypes ◽  
Polymerase Chain ◽  
A Site

In certain maize genotypes (nulls), β-glucosidase does not enter the gel and therefore cannot be detected on zymograms. Such genotypes were initially thought to be homozygous for a null allele at theglu1gene. We have shown that a β-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize β-glucosidases and forms large insoluble aggregates. To understand the mechanism of the β-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize β-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum β-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind. The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu50-Val145) and an extreme C-terminal region (Phe466-Ala512) together form the BGAF binding site on the enzyme surface. In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it inEscherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues. A data base search revealed that BGAF is a member of the jasmonite-induced protein family. Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice. Each of these repeat units is postulated to be involved in forming a site for binding to maize β-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays.

scholarly journals Ontogeny of erythropoietin gene expression in the sheep fetus: effect of dexamethasone at 60 days of gestation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 460-466
Author(s):  
GB Lim ◽  
K Jeyaseelan ◽  
EM Wintour
Keyword(s):  
Gene Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fetal Plasma ◽  
Liver And Kidney ◽  
Polymerase Chain ◽  
High Level ◽  
A Site ◽  
Sheep Fetus

We have used competitive reverse transcription and polymerase chain reaction (RT/PCR) to compare the levels of erythropoietin (Epo) mRNA in the liver and kidneys of the sheep fetus at 60, 80, 100, 130, and 140 days of gestation (term = 145 to 150 days). The effect of dexamethasone infusion in the ewe on Epo gene expression in the 60-day fetus was also investigated. Epo mRNA levels were highest at 60 days of gestation, the earliest age studied, in both liver and kidney. In the liver, Epo mRNA expression declined as gestation proceeded. Kidney Epo mRNA was maintained at a high level until 100 days of gestation, declining significantly in the 130-day fetus (P < .01). Treatment of ewes carrying 60-day fetuses with 0.76 mg/h dexamethasone for 48 hours resulted in a significant decrease in fetal plasma Epo values and Epo mRNA levels in both the liver and kidney. In the dexamethasone-treated fetuses, Epo mRNA in the liver was 52% of control values (P < .05), and in the kidney, 33% of control (P < .001). The results suggest that the kidney may play a more important role as a site of Epo synthesis in the early gestation sheep fetus than previously thought. Glucocorticoids may have a role in the regulation of Epo gene expression.

The Two mRNAS Encoding Rat Intestinal Alkaline Phosphatase Represent Two Distinct Nucleotide Sequences

1992 ◽  
Vol 38 (12) ◽  
pp. 2506-2509 ◽  
Author(s):  
M J Engle ◽  
D H Alpers
Keyword(s):  
Polymerase Chain Reaction ◽  
Alkaline Phosphatase ◽  
Duodenal Mucosa ◽  
Differential Regulation ◽  
Untranslated Regions ◽  
Terminal Sequence ◽  
Intestinal Alkaline Phosphatase ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Fat Feeding

Abstract Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.

Identification of a 4-base deletion in the gene in inherited intrinsic factor deficiency

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1515-1517 ◽  
Author(s):  
Fawwaz Yassin ◽  
Sheldon P. Rothenberg ◽  
Sreedhar Rao ◽  
Marilyn M. Gordon ◽  
David H. Alpers ◽  
...  
Keyword(s):  
Intrinsic Factor ◽  
Nucleotide Sequence Analysis ◽  
Coding Region ◽  
Chain Reaction ◽  
Exon 2 ◽  
Factor Deficiency ◽  
Schilling Test ◽  
Intrinsic Factor Deficiency ◽  
Polymerase Chain ◽  
A Site

Abstract A 4-base deletion has been identified in the coding region of the gene for gastric intrinsic factor (IF) in an 11-year-old girl with severe anemia and cobalamin (Cbl) deficiency. The bone marrow showed frank megaloblastic morphology, and the Schilling test indicated a failure to absorb Cbl that was corrected by coadministration of IF. Pentagastrin administration induced acid secretion, but the gastric juice lacked IF as determined by CbI binding, by fractionation of protein-bound CbI, and by immunoprecipitation with anti-IF antiserum. Individual exons were amplified by the polymerase chain reaction by using primers to the flanking intronic regions, and the nucleotide sequence analysis identified a 4-base deletion (c183_186delGAAT) spanning positions 104 to 107 in exon 2, resulting in premature termination of translation. This mutation also eliminates a site for Bst XI endonuclease and introduces a site for BsaBI for identifying this deletion in hereditary IF deficiency.

Abridged 5S rDNA units in sea beet (Beta vulgaris subsp. maritima)

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 352-354 ◽  
Author(s):  
Daniel J Turner ◽  
Terence A Brown
Keyword(s):  
Network Analysis ◽  
Beta Vulgaris ◽  
5S Rdna ◽  
Full Length ◽  
Rdna Repeat ◽  
Chain Reaction ◽  
Repeat Units ◽  
Unit Classes ◽  
Polymerase Chain ◽  
5S Gene

Amplification by polymerase chain reaction of the 5S rDNA repeat units of Beta vulgaris subsp. maritima resulted in a 350-bp product corresponding to the full-length 5S unit, but also revealed 4 abridged unit classes, each with a deletion that removed most of the spacer and 12–76 bp of the coding sequence. Each abridged type lacks at least 1 of the conserved elements involved in transcription of the 5S gene, and so appear to be nonfunctional. Network analysis revealed that the abridged units are evolving in the same manner as the full-length versions.Key words: 5S rDNA, Beta vulgaris subsp. maritima, network analysis, sea beet.

scholarly journals Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection

2019 ◽  
Vol 65 (6) ◽  
pp. 477-484 ◽  
Author(s):  
S.P. Radko ◽  
S.A. Lapa ◽  
A.V. Chudinov ◽  
S.A. Khmeleva ◽  
M.M. Mannanova ◽  
...  
Keyword(s):  
Chain Reaction ◽  
Dna Library ◽  
Aptamer Selection ◽  
Smad4 Protein ◽  
Dna Libraries ◽  
Polymerase Chain ◽  
Smad Binding Element ◽  
A Site ◽  
Oligonucleotide Sequence ◽  
Selection Of

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

scholarly journals Ontogeny of erythropoietin gene expression in the sheep fetus: effect of dexamethasone at 60 days of gestation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 460-466 ◽  
Author(s):  
GB Lim ◽  
K Jeyaseelan ◽  
EM Wintour
Keyword(s):  
Gene Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fetal Plasma ◽  
Liver And Kidney ◽  
Polymerase Chain ◽  
High Level ◽  
A Site ◽  
Sheep Fetus

Abstract We have used competitive reverse transcription and polymerase chain reaction (RT/PCR) to compare the levels of erythropoietin (Epo) mRNA in the liver and kidneys of the sheep fetus at 60, 80, 100, 130, and 140 days of gestation (term = 145 to 150 days). The effect of dexamethasone infusion in the ewe on Epo gene expression in the 60-day fetus was also investigated. Epo mRNA levels were highest at 60 days of gestation, the earliest age studied, in both liver and kidney. In the liver, Epo mRNA expression declined as gestation proceeded. Kidney Epo mRNA was maintained at a high level until 100 days of gestation, declining significantly in the 130-day fetus (P < .01). Treatment of ewes carrying 60-day fetuses with 0.76 mg/h dexamethasone for 48 hours resulted in a significant decrease in fetal plasma Epo values and Epo mRNA levels in both the liver and kidney. In the dexamethasone-treated fetuses, Epo mRNA in the liver was 52% of control values (P < .05), and in the kidney, 33% of control (P < .001). The results suggest that the kidney may play a more important role as a site of Epo synthesis in the early gestation sheep fetus than previously thought. Glucocorticoids may have a role in the regulation of Epo gene expression.

scholarly journals A novel DNA inversion causing severe hemophilia A

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3255-3261 ◽  
Author(s):  
JA Naylor ◽  
P Nicholson ◽  
Anne Goodeve ◽  
S Hassock ◽  
I Peake ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Hemophilia A ◽  
Severe Hemophilia ◽  
Chain Reaction ◽  
Abnormal Result ◽  
Extra Copy ◽  
Dna Inversion ◽  
Gene Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
A Site

Almost half of all cases of severe hemophilia A are caused by recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene. These inversions generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. They are routinely detected by a Bcl I Southern blot assay in which the sizes of two of the three normal hybridization bands are characteristically altered. However, atypical hybridization patterns have been observed, and this report describes the first detailed analysis of a hemophilia A patient with such a pattern. The abnormal result was found to be caused by a novel FVIII inversion involving an extra copy of int22h from a site only 70 to 200 kb telomeric of the FVIII gene. Polymerase chain reaction (PCR) allowed one of the inversion junctions to be analyzed, showing that the int22h sequence at this inversion junction was truncated. This patient and his novel inversion provide further evidence that int22h is associated with instability in Xq28.

Improved specificity ofTrypanosoma cruziidentification by polymerase chain reaction using an oligonucleotide derived from the amino-terminal sequence of a Tc24 protein

Parasitology ◽  
1995 ◽  
Vol 111 (5) ◽  
pp. 581-590 ◽  
Author(s):  
A. Taibi ◽  
A. Guevara-Espinoza ◽  
R. Schöneck ◽  
B. Yahiaoui ◽  
A. Ouaissi
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Value ◽  
Terminal Sequence ◽  
Chain Reaction ◽  
Blood Samples ◽  
Specific Diagnosis ◽  
Amino Terminal ◽  
Polymerase Chain ◽  
Amino Terminal Domain ◽  
Amino Terminal Sequence

SUMMARYIn the present study, the diagnostic value ofTrypanosoma cruzirecombinant protein (Tc24) was examined. Although antibodies against Tc24 were detected during natural and experimentalT. cruziinfections, specificity studies revealed that sera fromT. rangeli-infected mice also recognized to some extent Tc24 protein. In addition, sera from Tc24-immunized mice reacted against a 21 kDa polypeptide inT. rangeliextracts. Detailed analysis of the antibody response against 20—40 peptide localized in the Tc24 amino-terminal domain suggests that this sequence is not expressed byT. rangeli21 kDa antigen. Therefore, the PCR reaction using oligonucleotides corresponding to a 20–26 peptide clearly demonstrated the specificity of the oligoprobes forT. cruziidentification. Positive signals were also found when using blood samples fromT. cruzi-infected mice. Taken together, these results suggest that the PCR-based 20–26 assay may be useful in the specific diagnosis of Chagas' disease.

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