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.     

Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.     

Mammalian and Malaria Parasite Cyclase-associated Proteins Catalyze Nucleotide Exchange on G-actin through a Conserved Mechanism

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.     

Symplastic Solute Transport and Avocado Fruit Development: A Decline in Cytokinin/ABA Ratio is Related to Appearance of the Hass Small Fruit Variant

Studies on the effect of fruit size on endogenous ABA and isopentenyladenine(iP) in developing avocado (Persea americana Mill. cv. Hass)fruit revealed that ABA content was negatively correlated withfruit size whilst the iP/ABA ratio showed a linear relationshipwith increasing size of fruit harvested 226 d after full bloom.The effect of this change in hormone balance on the relationshipbetween symplastic solute transport and appearance of the smallfruit variant was examined following manipulation of the endogenouscytokinin (CK)/ABA ratio. Application of ABA caused seed coatsenescence and retarded fruit growth but these effects wereabsent in fruit treated with equal amounts of ABA plus iP. Thus,the underlying physiological mechanisms associated with ABA-inducedretardation of Hass avocado fruit growth appeared to be inextricablylinked to a decline in CK content and included: diminution ofmesocarp and seed coat plasmodesmatal branching, gating of mesocarpand seed coat plasmodesmata by deposition of electron densematerial in the neck region, abolishment of the electrochemicalgradient between mesocarp and seed coat parenchyma, and arrestof cell-to-cell chemical communication. (Received February 25, 1998; Accepted July 28, 1998)     

Full length L1 retroposons contain tRNA-like sequences near the 5' termini--hypothesis on the replication mechanism of retroposons

Retrotransposons replicate via a complex mechanism which depends on, among other things, the presence of long terminal repeats (LTRs) and a tRNA binding site just 3' of the 5' LTR. The LINES 1 (L1) family of sequences, which similar to retrotransposons in many other properties, represents a new class of retroposon which does not possess LTRs. However, we show here that the repetitive 5' motif associated with murine L1 elements contains a tRNA-like sequence in a location analogous to the position of the retro-transposon tRNA binding site. Although the repetition of such a 5' motif has only been found associated with murine L1 elements, we have found an analogous tRNA-like sequence near the 5' ends of the L1 elements from each of the other analyzed species for which the L1 family has been characterized, that is rat (L1Rr), human (L1Hs), drosophila (I element) and trypanosome (INGI). The conservation of this tRNA-like sequence near the 5' terminus of L1 elements from such diverse species suggests that it plays a functional role in the life of the L1 class of retroposon.     

Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell ‘hallmarks’. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.     

ImmunoTrack: the novel antibody-based technology for tracing in animal health

This paper describes a novel antibody-based livestock movement control tool and method of meat allocation, both in livestock husbandry as well as during the meat-processing chain. Immuno Track fulfills diverse prerequisites and meets regulatory demands which are substantial for a successful monitoring technology: (i) the induction of long-lasting antibody responses detectable onsite throughout the whole mast period of pigs, (ii) a single immunization injection with protein derivatives is sufficient to evoke a strong epitope-specific antibody response, and (iii) the complete degradation of the protein markers after the antibody response has been triggered in meatproducing animals such as cattle or pigs. There are diverse fields of application for the Immuno-Track marker technology, such as in quality meat programs, as compliance markers for animal vaccines or as a tool for verification of origin. Combination of this monitoring technology with the husbandry and identification databases for cattle and pigs within the European Community will lead to greater transparency in meat production, thereby regaining consumers' trust in concomitant structures of the meat-producing industry.