Bacteriophage T4 Head Morphogenesis II. Studies on the Maturation of Gene 49-Defective Head Intermediates

An investigation into the metabolic requirements for maturation of gene 49-defective heads indicated that adenosine triphosphate energy and continued deoxyribonucleic acid (DNA) but not ribonucleic acid synthesis were needed. The fate of DNA present at restrictive temperatures (41.5 C) in tsC9 (gene 49)-infected cells was also examined. After lysis of infected cells, the 12 to 32% deoxyribonuclease-resistant DNA associated with isolated gene 49-defective heads was found to be attached to a deoxyribonuclease-sensitive complex associated with the debris. Pulsechase experiments where (3)H-thymidine was used to label the DNA at 41.5 C suggested that more DNA from this pool was present in phage recovered after rescue of the gene 49 function than could be accounted for by the deoxyribonuclease-resistant portion. Further, when these experiments were repeated with an additional density shift ((15)N(13)C-glucose to (14)N(12)C-glucose), the DNA extracted from phage rescued at 10 min after the temperature shift-down was found to be 90% conserved. These results suggest a model whereby DNA packaging into capsid precursors is separated from DNA replication and the energy from DNA synthesis provides the driving force for packaging. Pulse-chase, temperature-shift experiments with E920g (gene 66) or E920g;tsC9 mutant-infected cells showed that gene (49, 66)-defective heads, which were isolated as small, isometric-shaped unfilled heads, were a precursor to "petite" phage. This suggests that the maturation process is independent of the size and shape of the head membrane. Similar experiments with the double mutant tsC9;amN120 indicate that gene 49-defective heads can also be filled in the absence of tails.     

Bacteriophage T4 head morphogenesis. VII. Terminal stages of head maturation.

Several aspects of the terminal stages of T4 head maturation were investigated using ts and am mutants blocked at single steps of the assembly pathway. We had previously found that cells infected with mutants of gene 13, e.g., tsN38 and amE609, accumulated both stable (10 to 20%)- and fragile (80%)-filled head precursors (Hamilton and Luftig, 1972). Here we showed the following for such gene 13-defective, mutant-infected cells. (i) Using thin-section analysis the pool of phage precursor structures observed under nonpermissive conditions was one-third of that observed when the cells were cultured under permissive conditions. (ii) In order for complete conversion of the precursors into viable phage to occur, there were apparent requirements of metabolic energy, protein, and DNA synthesis. (iii) The intracellular DNA pool under nonpermissive conditions exhibited a 50% distribution between 63S (mature size) and 200 S (concatenate size) DNA, with the latter DNA serving as a precursor pool. Further, this DNA pool when spread onto a protein monolayer exhibited a dispersed array of DNA, strands around a core, which was less dense than that found for the greater than 1,000S DNA concatenate isolated from gene 49-defective infected cells. (iv) When precuations were taken to stabilize the head precursors, such as lysis of the cells into glutaraldehyde, there was a 30% increase in the yield of 1,200S filled heads. Correlating these results and previous results concerning gene 49-defective unfilled heads, we propose that there are several forms of gene 13 fragile head precursors which serve as intermediates between gene 49 unfilled heads and gene 13 stable filled heads. We cannot, however, rule out the possibility that all gene 13-defective heads represent a single class of unstable particles, which decay slowly. In either case, we have shown that gene 13-defective particles are unstable to some degree inside the cell and are highly unstable outside the cell; yet all particles can still be efficiently converted to phage in vivo.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA.

With the exception of mutants in gene 49, all mutants in phage T4 defective in the process of head filling accumulate a normal replicative DNA intermediate of 200S. Mutants in gene 49 produce a very fast-sedimenting (VFS) DNA with s values of greater than 1,000S. The intracellular development of the VFS-DNA generated in gene 49-defective phage-infected cells was followed by sedimentation analysis of crude lysates on neutral sucrose gradients. It was observed that the production of a 200S replicative intermediate is one step in the development of VFS-DNA. After restoring permissive conditions the development of the VFS-DNA can be reversed, but the 200S form is not regenerated under these conditions. The process of head filling can take place from the VFS-DNA under permissive conditions. From the absence of other components in the VFS-DNA complexes, its high resistance to shearing, its resistance against the attack of the single-strand-specific nuclease S1, and from its appearance in the electron microscope, a complex structure of tightly packed DNA is inferred. The demonstration by the electron microscope of branched DNA structures sometimes closely related to partially filled heads is taken in support of the idea that the process of head filling in gene 49-defective phage-infected cells is blocked by some steric hindrance in the DNA. In light of these results, the role of gene 49 is discussed as a control function for the clearance of these structures. A fixation procedure for cross-linking of gene 49-defective heads to the VFS-DNA allowed us to study progressive stages in the process of head filling. Electron microscopic evidence is presented which suggests that during the initial events the DNA accumulates in the vertexes of the head.     

Bacteriophage T4 Head Morphogenesis III. Some Novel Properties of Gene 13-Defective Heads

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.     

Genetic control of whisker antigen of bacteriophage T4D

An antigenic component of T4 whiskers (short fibrils located in the region of the head—tail junction) has been reported to be under the control of gene 49 (Yanagida & Ahmad-Zadeh, 1970; Yanagida, 1972). This was based on immunological evidence using antiserum to particles of T4D adsorbed with gene 49-defective extract made with the mutant amE727. The latter phage, however, is shown here to be a double mutant bearing amber mutations in gene 49 and another gene, herein referred to as wac (whisker antigen control gene). Gene wac maps in the general region of gene 16. Evidence is presented indicating that the whisker antigen is under the control of wac and not gene 49. In wac-defective infections phage are produced that lack a protein. This protein appears by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels to be the major component of the antigen.The tail fibers of wac-defective bacteriophage are in an open configuration under conditions in which those of wild-type phage are folded alongside the tail. Thus, the wac gene may have a role in the regulation of tail-fiber configuration.     

Bacteriophage T4D Head Morphogenesis. VIII. DNA-Protein Associations in Intermediate Head Structures That Accumulate in Gene 49? Mutant-Infected Cells

We have utilized the gene 49⁻ mutant-infected cells of bacteriophage T4D to accumulate large numbers of nucleic acid-protein intermediate head structures. These heads were used as substrates for experiments in the investigations of the mechanism of DNA packaging. Specifically, we have examined: (i) the susceptibility of the DNA in these structures to digestion by a variety of nucleases after a series of increasing temperature pulses from 25 to 100°C, (ii) the physicochemical characteristics of the DNA inside these heads, and (iii) the mechanism by which proteins are displaced from the interior of the head after treatment with basic proteins. We isolated DNA from these gene 49⁻ heads by use of gradient centrifugation procedures. The DNA had a molecular weight of 8 × 10⁶ and a density of 1.697 ± 0.005 g/cm³, and it contained a short resistant fraction (SRF) which, when associated with the gene 49⁻ heads, exhibited AT-protected regions that were not susceptible to micrococcal nuclease digestion. Such a fraction may contain pieces which are important in the initial association of the DNA with the prohead. Exposure of the gene 49⁻ intermediate capsid structures to basic proteins, such as bovine trypsin inhibitor, lysozyme, and l-polylysine-70, caused a displacement of an amorphous-appearing structure which may be a complex of the gene 49⁻ DNA and interior components of the capsid (e.g., internal proteins, polyamines). Our general conclusion is that in the gene 49⁻ intermediate head structures which are only partly filled with DNA, this DNA is held inside the head by strong electrostatic linkages with interior polypeptides and polyamines.     

Bacteriophage T4 Head Maturation: Release of Progeny DNA from the Host Cell Membrane

We have presented a new approach to studying bacteriophage T4 head maturation. Using a modified M-band technique, we have shown that progeny deoxyribonucleic acid (DNA) was synthesized on the host cell membrane throughout infection. This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31, 16, 17 and 49, known to be necessary for normal head formation. Gene products 2, 4, 50, 64, 65, 13 and 14, also involved in head morphogenesis were not required to detach progeny DNA from the membrane; the presence of the phage tail and tail fibers also was not required. DNA was released in the form of immature heads and initially was sensitive to deoxyribonuclease (DNase). Conversion to DNase resistance followed rapidly. The amount of phage precursors present at the time of DNA synthesis determined the time of onset and detachment rate of DNA from the M band as well as the kinetics by which the detached DNA become DNase resistant.     

Genetic Control of Capsid Length in Bacteriophage T4 I. Isolation and Preliminary Description of Four New Mutants

Four new mutants are described whose phenotypic expression affects the length of the head of bacteriophage T4D. All mutants produce some phenotypically normal phage particles. Mutant pt21-34 also produces at least two size classes of phage particle which have heads that are shorter than normal. The other three mutants, ptg19-2, ptg19-80, and ptg191, produce, in addition to phages with normal and with shorter-than-normal heads, giant phages with heads from 1.5 to at least 10 times the normal length. All mutations are clustered near gene 23. Giant phage particles have the following properties: they are infectious and contain and inject multiple genomes as a single continuous bihelical DNA molecule of greater-than-unit length. Their frequency, relative to the total plaque-former population, increases late in the infectious cycle. They have a normal diameter, variable length, and a buoyant density range in CsCl from equal to slightly greater than that of normal phage. The arrangement of capsomers is visible in the capsids, which are composed of cleaved gene 23 protein.     

Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants

Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

INVESTIGATION OF SECONDARY STRUCTURES AND MACROMOLECULAR INTERACTIONS IN BACTERIOPHAGE P22 BY LASER RAMAN SPECTROSCOPY

Laser Raman spectra of the DNA bacteriophage P22 and of its precursor particles and related structures have been obtained using 514.5-nm excitation. The spectra show that P22 DNA exists in the B form both inside of the phage head and after extraction from the phage. The major coat protein (gp5) contains a secondary structure composed of 18% α-helix, 20% β-sheet and 62% irregular conformations. The scaffolding protein (gp8) in the phage prohead is substantially richer than gp5 in α-helical content. Among the amino acid residues which give prominent Raman lines, the spectra show that tryptophans are exposed to solvent and most tyrosines are hydrogen bonded to positive donor groups. The above features of phage DNA and protein structures are nearly invariant to changes in temperature up to 80°C, indicating a remarkable thermal stability of the phage head and its encapsulated DNA.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

Functional empty capsid precursors produced by lambda mutant defective for late lambda DNA replication.

This report described lambda phage morphogenesis in a mutant system in which the normal pathways for late phage DNA (concatemer) synthesis are blocked and early (monomeric circular) DNA replication products accumulate. As shown earlier (Dawson et al., 1975) under these conditions, late proteins are synthesized and assembled into headlike structures. These structures that accumulate in the mutant are empty, suggesting the monomeric circular DNA molecules cannot be encapsulated. The present results show that crude extracts of induced lysogens of the mutant contain the complementation activities of preheads (the empty precursors to DNA-filled heads), tails, and DNA terminigenerating protein(s). Sucrose gradients of these crude extracts yield fractions containing prehead activity in relative amounts expected from the concentration of late proteins and empty structures. Furthermore, the proteins present in these fractions coelectrophorese with the known capsid proteins of preheads, and empty structures that look like preheads are observed in electron microscope examination of samples from the fractions. Based on our biological, biochemical, and electron microscope analyses, we conclude that the empty structures that accumulate in the induced lysogen of the mutant are normal preheads, which could become filled phage heads if DNA of the appropriate structure (i.e., "late DNA") were available.     

Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.     

Comparative efficacy of long-acting bronchodilators for COPD - a network meta-analysis

Background

Clinicians are faced with an increasingly difficult choice regarding the optimal bronchodilator for patients with chronic obstructive pulmonary disease (COPD) given the number of new treatments. The objective of this study is to evaluate the comparative efficacy of indacaterol 75/150/300 μg once daily (OD), glycopyrronium bromide 50 μg OD, tiotropium bromide 18 μg/5 μg OD, salmeterol 50 μg twice daily (BID), formoterol 12 μg BID, and placebo for moderate to severe COPD.

Methods

Forty randomized controlled trials were combined in a Bayesian network meta-analysis. Outcomes of interest were trough and post-dose forced expiratory volume in 1 second (FEV₁), St. George’s Respiratory Questionnaire (SGRQ) score and responders (≥4 points), and Transition Dyspnea Index (TDI) score and responders (≥1 point) at 6 months.

Results

Indacaterol was associated with a higher trough FEV₁ than other active treatments (difference for indacaterol 150 μg and 300 μg versus placebo: 152 mL (95% credible interval (CrI): 126, 179); 160 mL (95% CrI: 133, 187)) and the greatest improvement in SGRQ score (difference for indacaterol 150 μg and 300 μg versus placebo: -3.9 (95% CrI -5.2, -2.6); -3.6 (95% CrI -4.8, -2.3)). Glycopyrronium and tiotropium 18 μg resulted in the next best estimates for both outcomes with minor differences (difference for glycopyrronium versus tiotropium for trough FEV₁ and SGRQ: 18 mL (95% CrI: -16, 51); -0.55 (95% CrI: -2.04, 0.92).

Conclusion

In terms of trough FEV₁ and SGRQ score indacaterol, glycopyrronium, and tiotropium are expected to be the most effective bronchodilators.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Improved Methods of Carnivore Faecal Sample Preservation,DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

Background

Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase.

Methods

We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval.

Results

Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively.

Conclusions

Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.