Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.     

Nucleotide Sequence Surrounding the Locus Marker D21S246 on Human Chromosome 21

Most ofthe human Not I linking clones identified to date areconsidered to be derived from CpG islands because ofthe recognitionsequence of this enzyme, and CpG islands have been reportedto be located around the 5' regions of genes. As a pilot study,we determined the complete nucleotide sequence (41,924 bp) ofa human cosmid clone (LL21NC02Q7A10) containing the marker D21S246originating from a Not I linking clone. As a result of sequenceanalysis, we successfully mapped and revealed the genomic genestructure for KIAA0002 previously reported as a cDNA clone.This gene consists of 15 exons and was shown to exist at theD21S246 locus on human chromosome 21q21.3–q22.1. Theseresults demonstrated that genomic marker-anchored DNA sequencingis a useful approach for the human genome project.     

Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5′-AT/TAAT-3′

Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5-AT/TAAT-3 generating 5 dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of VspI, a restriction enzyme isolated from Vibrio sp.     

Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Investigations of mechanisms of resistance to bipyridyl herbicides in Arctotheca calendula (L.) Levyns

The mechanism of resistance to diquat and paraquat was investigated in a bipyridyl-herbicide-resistant biotype of Arctotheca calendula (L.) Levyns. No differences were observed in the interactions of these herbicides with Photo-system I, the active site, in thylakoids isolated from resistant and susceptible biotypes. Likewise, absorption of herbicide through the cuticle and gross translocation were identical in plants of the two biotypes. Foliar application of either 25 g ha⁻¹ diquat or 200 g ha⁻¹ paraquat rapidly inhibited CO₂-dependent O₂ evolution of leaf segments of the susceptible biotype. O₂ evolution of leaf segments of the resistant biotype was less affected by these treatments. Fluorescence imaging was used to observe visually, as fluorescence quenching, the penetration of herbicide to the active site. These experiments demonstrated that diquat appears at the active site more slowly in the resistant biotype compared to the susceptible biotype. HCO₃-dependent O₂ evolution of thin leaf slices was less inhibited by diquat in the resistant biotype than in the susceptible biotype. The mechanism of resistance to the bipyridyl herbicides in this biotype of A. calendula is not a result of changes at the active site, decreased herbicide absorption or decreased translocation, but appears to be due to reduced herbicide penetration to the active site.     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

Some improvements in the quality of NAA procedures and the reliability of the results

After considering the need for quality control in NAA, the concept of quality in NAA procedures themselves is discussed, and some important factors identified. Two approaches to improve quality are then described in more detail. The first concerns the unique ability of NAA using different isotopic reactions and different modes (INAA/RNAA) to provide independent data sets in the same laboratory, thus allowing internal validation or crosschecking. The second discusses the need for chemical yield measurements in RNAA and the advantages of the radioisotopic tracer technique. Some recent advances and further possibilities for this use of tracers are listed.     

Accumulation of cesium and radiocesium in forest litter in selected regions of poland and its influence on litter-to-mushroom transfer factor

The aim of this work was to check whether the stable cesium content in forest litter affects the value of radiocesium from litter-to-mushroom transfer factorTf or not. Total cesium in litter, measured by AAS, varied from 0.1–2.7 μg/g. These data, combined with earlier results for mushrooms, showed no simple correlation forTf. More complex relationships provided very high correlation coefficients, but their validity needs further investigation.