Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP side of γ-Cornephage

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.     

Somatic polyploidy in extraembryonic membranes of the snake,Elaphe guttata

Summary Video-densitometric DNA measurements of Feulgenstained tissues of 42 day old eggs of the corn snake,Elaphe g. guttata (Columbridae, Serpentes), revealed a basic DNA content of 2C=2.17 pg, with somatic polyploidy in the allantois, the chorioallontois, the yolk sac, and other extraembryonic membranes. The maximum value determined was 128C (in binucleate cells 2×128C) at the distal pole of the egg. This is the first report of somatic polyploidy in a snake, and one of the first in reptiles in general.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

EVIDENCE FOR A MOSAIC HYBRID ZONE IN THE GRASS SHRIMP PALAEMONETES KADIAKENSIS (PALAEMONIDAE) AS REVEALED BY MULTIPLE GENETIC MARKERS

Molecular techniques provide powerful tools for studying the geographic structure of hybrid zones and the dynamics of gene exchange between incipient species. We examined allozyme variation at five loci (PGM, GPI, MDH-1, MDH-2, and LDH) for 27 populations of Palaemonetes kadiakensis from the central, coastal, and eastern regions of Texas. Central Texas populations of P. kadiakensis exhibited highly significant linkage disequilibrium and departures from Hardy-Weinberg genotype proportions. In populations with linkage disequilibrium, allelic differences at GPI defined two types of P. kadiakensis, designated A and B. Both types existed in central Texas with little or no evidence of interbreeding, whereas the populations from all other localities showed complete introgression of type B alleles into the type A gene pool. We also examined ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) variation in a subset of populations, chosen to cover a range of geographic locations and levels of linkage disequilibrium. Two groups of mtDNA haplotypes and two restriction fragment patterns for the rDNA corresponded to allozyme type A and B individuals in populations exhibiting linkage disequilibrium. In populations with ongoing hybridization, all hybrid animals (N= 15) exhibited type A mtDNA. Exhibition of type A mtDNA indicated that type A females had mated successfully with type B males, but type B females had not mated successfully with type A males. Genotype distributions suggest reduced reproduction by hybrid offspring in central Texas populations. These patterns are consistent with a mosaic model of hybrid zone dynamics.