Bacteriophage burst size during multiple infections

A significant positive correlation was observed between multiplicity of infection and burst size of mycobacteriophage 13. During multiple infections, the average contribution of each infecting phage to the burst size was inversely correlated with multiplicity of infection even when bacterial resources were not limiting. We conclude that the efficiency of phage-coded functions rather than the extent of bacterial resources determines the burst size

Transcriptional specificity after mycobacteriophage I3 infection

Transcriptional regulation following mycobacteriophage I3 infection has been investigated. For this purpose, RNA polymerase mutants (rif) of host bacterium, Mycobacterium smegmatis have been isolated and characterised. Phage growth inrifs and rifr cells in presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies onin vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infected Mycobacterium smegmatis have been purified to homogeniety. The enzyme purification was accomplished by a rapid procedure utilising affinity chromatography on rifampicin-Sepharose columns. Subunit structure analysis of the purified RNA polymerase from uninfected and phage-infected cells showed the presence of α ,β , β ' and σ subunits similar to the other prokaryotic RNA polymerases. In addition, a polypeptide of 79, 000 daltons was associated with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage 13 DNA was the preferred template for the modified RNA polymerase isolated from infected cells which may account for the transcriptional switch required for phage development

DNA polymerase α -primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit

Isolation and characterization of DNA polymerase ε from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'→5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue

Analysis of nuclear proteins from silk glands of Bombyx mori

A gentle method for the isolation of nuclei from developing silk glands of Bombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development

DNA polymerase-delta from the silk glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated

Requirement for calcium ions in mycobacteriophage I3 DNA injection and propagation

Ca2+ ions are absolutely necessary for the propagation of mycobacteriophage I3 in synthetic medium. These ions are required for successful infection of the host and during the entire span of the intracellular development of the phage. A direct assay of the phage DNA injection using 32[p] labelled phage, shows that Ca2+ ions are necessary for the injection process. The injection itself is a slow process and takes 15 min to complete at 37°. The bacteria infected in presence of Ca2+ tend to abort if the ions are subsequently withdrawn from the growth medium. The effect of calcium withdrawal is maximally felt during the early part of the latent period; however, later supplementation of Ca2+ ions salvage phage production and the mature phage progeny appear after a delayed interval, proportional to the time of addition of Ca2+

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) from Mycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg2+, polyamines significantly stimulate the reaction. Of the cations tested, only Mn2+, Co2+ and Ca2+ can partially substitute for Mg2+ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyltRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KCl on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5-9.0 at 1.2 mM Mg2+ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg2+ is also prevntedvby spemine

Growth of Mycobacterium smegmatis in minimal and complete media

The growth patterns of Mycobacterium smegmat is SN2 in a minimal medium and in nutrient broth have been compared. The growth was monitored by absorbancy (Klett readings), colony forming units, wet weight and content of DNA, RNA and protein. During the early part of the growth cycle, the bacteria had higher wet weight and macromolecular content in nutrient broth than in minimal media. During the latter half of the growth cycle however, biosynthesis stopped much earlier in nutrient broth and the bacteria had a much lower content of macromolecules than in the minimal medium. In both the media, a general pattern of completing biosynthesis rapidly in the initial phase and a certain amount of cell division at a later time involving the distribution of preformed macromolecules was seen. The possible adaptive significance of this observation has been discussed

Confocal Microscopy: a Powerful Tool for Biological Research

Conventional light microscopy allows the observation of living as well as fixed cells and tissues to generate two-dimensional images. The out-of-focus information often obscures the ultrastructural details, especially in thick specimens with overlapping structures. The earliest available light microscopy visualized the objects in hydrated state in two-dimensions during their temporal development. The emergence of electron microscopy (EM) provided superb resolution of ultrastructural details, but it was applicable only for objects in the dehydrated state and thereby potentially introducing handling artifacts. The usefulness of optical methods, however, has been limited by the poor depth discrimination. Often, the fluorescence and reflectance images are severely degraded by the scattered- or emitted-light from tissue structures outside the plane of focus. These limitations have been partially overcome by video image processing and deconvolution. Laser scanning confocal microscopy (LSCM) overcomes the above difficulties and produces improved light microscopic images of fixed as well as living cells and tissues. In terms of resolution of the image, the confocal microscope occupies a position in between the light and electron microscopes. Confocal microscope generates information from a well-defined optical section rather than from the entire specimen, thereby eliminating the out-of-focus glare and increasing the contrast, clarity and detection sensitivity. Optical sectioning is noninvasive and less time consuming compared to reconstruction algorithms to give 3-D images. Optical sectioning is achieved not only in the xy plane (perpendicular to the optical axis of the microscope) but also vertically in the xz or yz plane (parallel to the optical axis). With vertical sectioning, cells are scanned laterally (x or y axis) as well as in depth (z axis). Stacks of optical sections taken at successive focal plane (known as z series) are then reconstructed to generate a 3-D version of the specimen. The 3-D image can be directly visualized where each data point represents the quantity of specific contrast parameter used at a certain point in space. The image processing can be additionally used to enhance the confocal images. It is widely used in the fluorescence mode for different specimens and in bright field reflection mode for objects of different forms