Morphogenesis of Mammalian Endoplasmic Reticulum and Golgi Apparatus throughout the Cell Cycle

The endoplasmic reticulum (ER) and the Golgi apparatus are organelles that produce, modify and transport proteins and lipids and regulate Ca2+ environment within cells. Structurally they are composed of sheets and tubules. Sheets may take various forms: intact, fenestrated, single or stacked. The ER, including the nuclear envelope, is a single continuous network, while the Golgi shows only some level of connectivity. It is often unclear, how different morphologies correspond to particular functions. Previous studies indicate that the structures of the ER and Golgi are dynamic and regulated by fusion and fission events, cytoskeleton, rate of protein synthesis and secretion, and specific structural proteins. For example, many structural proteins shaping tubular ER have been identified, but sheet formation is much more unclear. In this study, we used light and electron microscopy to study morphological changes of the ER and Golgi in mammalian cells. The proportion, type, location and dynamics of ER sheets and tubules were found to vary in a cell type or cell cycle stage dependent manner. During interphase, ER and Golgi structures were demonstrated to be regulated by p37, a cofactor of the fusion factor p97, and microtubules, which also affected the localization of the organelles. Like previously shown for the Golgi, the ER displayed a tendency for fenestration and tubulation during mitosis. However, this shape change did not result in ER fragmentation as happens to Golgi, but a continuous network was retained. The activity of p97/p37 was found to be important for the reassembly of both organelles after mitosis. In EM images, ER sheet membranes appear rough, since they contain attached ribosomes, whereas tubular membranes appear smooth. Our studies revealed that structural changes of the ER towards fenestrated and tubular direction correlate with loss of ER-bound ribosomes and vice versa. High and low curvature ER membranes have a low and high density of ribosomes, respectively. To conclude, both ER and Golgi architecture depend on fusion activity of p97/p37. ER morphogenesis, particularly of the sheet shape, is intimately linked to the density of membrane bound ribosomes.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Studies on the pro-oxidative properties and neurotoxic potential of Angeli's salt-derived nitroxyl (HNO/NO-)

The biological function of nitric oxide and its oxidized forms has received a great deal of attention over the past two decades. However much less attention has been focused on the reduced nitric oxide, nitroxyl (HNO). Unlike NO, HNO is highly reactive species and thus it needs to be generated by using donor compounds under experimental conditions. Currently there is only one donor available, Angeli s salt, which releases HNO in a controlled fashion under pysiological conditions. Prior studies have shown the pro-oxidative and cytotoxic potential of Angeli s salt compared to NO donors. The high reactivity of HNO with cysteine thiols is considered to form the biochemical basis for its unique properties compared to other nitrogen oxides. Such thiol modification cold result in disturbances of vital cellular functions and subsequently to death of disturbance sensitive cells, such as neurons. Therefore modification of proteins and lipids was studied in vitro and the potential neurotoxicity was studied in vivo by local infusion of Angeli s salt into the rat central nervous system. The results show that under aerobic in vitro conditions, HNO can, subsequent to autoxidation, cause irreversible oxidative modification of proteins and lipids. These effects are not however seen in cell culture or following infusion of Angeli s salt directly into the rat central nervous tissue likely due to presence of lower oxygen and higher thiol concentration. However, due to high reactivity with thiols, HNO can cause irreversible inactivation of cysteine modification sensitive enzymes such as cysteine proteases papain in vitro and cathepsin B in cell culture. Furthermore it was shown that infusion of HNO releasing Angeli s salt into the rat central nervous system causes necrotic cell death and motor dysfunction following infusion into the lumbal intrathecal space. In conclusion, the acute neurotoxic potential of Angeli s salt was shown to be relatively low, but still higher compared to NO donors. HNO was shown to affect numerous cellular processes which could result in neurotoxicity if HNO was produced in vivo.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

Biochemical and structural properties of Potato virus A VPg

The potato virus A (PVA) genome linked protein (VPg) is a multifunctional protein that takes part in vital infection cycle events such as replication and movement of the virus from cell to cell. VPg is attached to the 5´ end of the genome and is carried in the tip structure of the filamentous virus particle. VPg is also the last protein to be cleaved from the polyprotein. VPg interacts with several viral and host proteins and is phosphorylated at several positions. These features indicate a central role in virus epidemiology and a requirement for an efficient but flexible mechanism for switching between different functions. -- This study examines some of the key VPg functions in more detail. Mutations in the positively charged region from Ala38 to Lys44 affected the NTP binding, uridylylation, and in vitro translation inhibition activities of VPg, whereas in vivo translation inhibition was not affected. Some of the data generated in this study implicated the structural flexibility of the protein in functional activities. VPg lacks a rigid structure, which could allow it to adapt conformationally to different functions as needed. A major finding of this study is that PVA VPg belongs to the class of ´intrinsically disordered proteins´ (IDPs). IDPs are a novel protein class that has helped to explain the observed lack of structure. The existence of IDPs clearly shows that proteins can be functional and adapt a native fold without a rigid structure. Evidence for the intrinsic disorder of VPg was provided by CD spectroscopy, NMR, fluorescence spectroscopy, bioinformatic analysis, and limited proteolytic digestion. The structure of VPg resembles that of a molten globule-type protein and has a hydrophobic core domain. Approximately 50% of the protein is disordered and an α-helical stabilization of these regions has been hypothesized. Surprisingly, VPg structure was stabilized in the presence of anionic lipid vesicles. The stabilization was accompanied by a change in VPg structure and major morphological modifications of the vesicles, including a pronounced increase in the size and appearance of pore or plaque like formations on the vesicle surface. The most likely scenario seems to be an α-helical stabilization of VPg which induces formation of a pore or channel-like structure on the vesicle surface. The size increase is probably due to fusion or swelling of the vesicles. The latter hypothesis is supported by the evident disruption of the vesicles after prolonged incubation with VPg. A model describing the results is presented and discussed in relation to other known properties of the protein.

Microfiltration in cheese and whey processing

Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.

Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

Deriving structural information from non-coding ribonucleic acids by mass spectrometry

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

DNA Packaging and Host Cell Lysis: Late Events in Bacteriophage PRD1 Infection

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.