A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS-polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N-terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base-pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane-spanning region with a predicted -helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.     

Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota

The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction

ESAT-6 (the 6 kDa early secreted antigenic target) protein species in short-term culture filtrate of Mycobacterium tuberculosis were separated in a 4-5 narrow range pI gradient two-dimensional gel electrophoresis (2-DE). Eight ESAT-6 protein species were analyzed in detail by peptide mass fingerprinting matrix-assisted laser desorption/ionization-mass spectrometry as well as by electrospray ionization-tandem mass spectrometry. An N-terminal Thr acetylation was identified in four species and a C-terminal truncation was identified in two species. In 2-DE blot overlay assays, the recombinant 10 kDa culture filtrate protein (CFP10) discriminated N-terminal acetylated and nonacetylated ESAT-6 by differential interaction, whereas removal of the C-terminal 11 residues of ESAT-6 had no effects thereon. This example shows that the access to the protein species level can be a prerequisite to understand regulation of protein-protein interaction.     

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.