Presence of haemocyte-like cells in coccinellid reflex blood

Abstract.  Contrary to current assumptions, the reflex blood of two-spot ladybirds, Adalia bipunctata , and seven-spot ladybirds, Coccinella septempunctata , contains haemocyte-like cells. Furthermore, DNA can be extracted and amplified from coccinellid reflex blood, confirming the presence of haemocyte-like cells and demonstrating a nondestructive method of DNA extraction.     

Response of muscle-based biochemical condition indices to short-term variations in food availability in post-flexion reared sea bass Dicentrarchus labrax (L.) larvae

Six condition indices based on RNA, total soluble protein and two metabolic enzymes [lactate dehydrogenase (LDH) and citrate synthase (CS)] were analysed in muscle tissue of individual larvae, post-flexion reared sea bass Dicentrarchus labrax using DNA and total soluble protein as standards for size. In addition, the effect of 2 days of food deprivation on the cell proliferation rates was assessed. The RNA:DNA best reflected short-term changes in feeding conditions. If standardized by DNA content, LDH activity was a better indicator of condition than any other index but RNA:DNA. Further, the analysis of cell proliferation rates in muscle from 26 day-old larvae proved useful in distinguishing continuously fed larvae from individuals subjected to 2 days of fast.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

Neutral mutations and repetitive DNA

We have previously shown that computer simulations of processes that generate selectively advantageous changes together with random duplications and deletions give rise to genomes with many different genes embedded in a large amount of dispensable DNA sequence. We now explore the consequences of neutral changes on the evolution of genomes. We follow the consequences of sequence divergences that are neutral when they occur in dispensable sequences or extra copies of genes present in multigene families. We find that when divergence occurs at about the same frequency as duplication/deletion events, genomes carry repetitive sequences in proportion to their size. Inspection of the genomes as they evolved showed that multigene families were generated by relatively recent duplications of single genes and so would be expected to be highly homogeneous.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine